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The highly reactive dicarbonyl methylglyoxal (MGO) is mainly formed as byproduct of glycolysis. Therefore, high blood glucose levels determine increased MGO accumulation. Nonetheless, MGO levels are also increased as consequence of the ineffective action of its main detoxification pathway, the glyoxalase system, of which glyoxalase 1 (Glo1) is the rate-limiting enzyme. Indeed, a physiological decrease of Glo1 transcription and activity occurs not only in chronic hyperglycaemia but also with ageing, during which MGO accumulation occurs. MGO and its advanced glycated end products (AGEs) are associated with age-related diseases including diabetes, vascular dysfunction and neurodegeneration. Endothelial dysfunction is the first step in the initiation, progression and clinical outcome of vascular complications, such as retinopathy, nephropathy, impaired wound healing and macroangiopathy. Because of these considerations, studies have been centered on understanding the molecular basis of endothelial dysfunction in diabetes, unveiling a central role of MGO-Glo1 imbalance in the onset of vascular complications. This review focuses on the current understanding of MGO accumulation and Glo1 activity in diabetes, and their contribution on the impairment of endothelial function leading to diabetes-associated vascular damage.

Aims/hypothesis Potentiation of glucose-induced insulin secretion is the main mechanism of exenatide (EXE) antidiabetic action, however, increased glucose utilization by peripheral tissues has been also reported. We here studied the effect of EXE on glucose uptake by skeletal muscle cells. Methods 2-deoxy-glucose (2DG) uptake and intracellular signal pathways were measured in rat L6 skeletal muscle myotubes exposed to 100 nmol/l EXE for up to 48 h. Mechanisms of EXE action were explored by inhibiting AMPK activity with compound C (CC, 40 μmol/l) or siRNAs (2 μmol/l). Results Time course experiments show that EXE increases glucose uptake up to 48 h achieving its maximal effect, similar to that induced by insulin, after 20 min (2- vs 2.5-fold-increase, respectively). Differently from insulin, EXE does not stimulate: (i) IR β-subunit- and IRS1 tyrosine phosphorylation and binding to p85 regulatory subunit of PI-3kinase; (ii) AKT activation; and (iii) ERK1/2 and JNK1/2 phosphorylation. Conversely, EXE increases phosphorylation of α-subunit of AMPK at Thr172 by 2.5-fold (p < 0.01). Co-incubation of EXE and insulin does not induce additive effects on 2DG-uptake. Inhibition of AMPK with CC, and reduction of AMPK protein expression by siRNA, completely abolish EXE-induced 2DG-uptake. Liraglutide, another GLP-1 receptor agonist, also stimulates AMPK phosphorylation and 2DG-uptake. Moreover, EXE stimulates 2DG-uptake also by L6 myotubes rendered insulin-resistant with methylglyoxal. Finally, EXE also induces glucose transporter Glut-4 translocation to the plasma membrane. Conclusions/interpretation In L6 myotubes, EXE and liraglutide increase glucose uptake in an insulin-independent manner by activating AMPK.

Dicarbonyl stress occurs when dicarbonyl metabolites (i.e., methylglyoxal, glyoxal and 3-deoxyglucosone) accumulate as a consequence of their increased production and/or decreased detoxification. This toxic condition has been associated with metabolic and age-related diseases, both of which are characterized by a pro-inflammatory and pro-oxidant state. Methylglyoxal (MGO) is the most reactive dicarbonyl and the one with the highest endogenous flux. It is the precursor of the major quantitative advanced glycated products (AGEs) in physiological systems, arginine-derived hydroimidazolones, which accumulate in aging and dysfunctional tissues. The aging process is characterized by a decline in the functional properties of cells, tissues and whole organs, starting from the perturbation of crucial cellular processes, including mitochondrial function, proteostasis and stress-scavenging systems. Increasing studies are corroborating the causal relationship between MGO-derived AGEs and age-related tissue dysfunction, unveiling a previously underestimated role of dicarbonyl stress in determining healthy or unhealthy aging. This review summarizes the latest evidence supporting a causal role of dicarbonyl stress in age-related diseases, including diabetes mellitus, cardiovascular disease and neurodegeneration.

Aims/hypothesis Insulin exerts a direct action on vascular cells, thereby affecting the outcome and progression of diabetic vascular complications. However, the mechanism through which insulin signalling is impaired in the endothelium of diabetic individuals remains unclear. In this work, we have evaluated the role of the AGE precursor methylglyoxal (MGO) in generating endothelial insulin resistance both in cells and in animal models. Methods Time course experiments were performed on mouse aortic endothelial cells (MAECs) incubated with 500 μmol/l MGO. The glyoxalase-1 inhibitor S - p -bromobenzylglutathione-cyclopentyl-diester (SpBrBzGSHCp2) was used to increase the endogenous levels of MGO. For the in vivo study, an MGO solution was administrated i.p. to C57BL/6 mice for 7 weeks. Results MGO prevented the insulin-dependent activation of the IRS1/protein kinase Akt/endothelial nitric oxide synthase (eNOS) pathway, thereby blunting nitric oxide (NO) production, while extracellular signal-regulated kinase (ERK1/2) activation and endothelin-1 (ET-1) release were increased by MGO in MAECs. Similar results were obtained in MAECs treated with SpBrBzGSHCp2. In MGO- and SpBrBzGSHCp2-exposed cells, inhibition of ERK1/2 decreased IRS1 phosphorylation on S616 and rescued insulin-dependent Akt activation and NO generation, indicating that MGO inhibition of the IRS1/Akt/eNOS pathway is mediated, at least in part, by ERK1/2. Chronic administration of MGO to C57BL/6 mice impaired whole-body insulin sensitivity and induced endothelial insulin resistance. Conclusions/interpretation MGO impairs the action of insulin on the endothelium both in vitro and in vivo, at least in part through an ERK1/2-mediated mechanism. These findings may be instrumental in developing novel strategies for preserving endothelial function in diabetes.

Metabolic and/or endocrine dysfunction of the white adipose tissue (WAT) contribute to the development of metabolic disorders, such as Type 2 Diabetes (T2D). Therefore, the identification of products able to improve adipose tissue function represents a valuable strategy for the prevention and/or treatment of T2D. In the current study, we investigated the potential effects of dry extracts obtained from Citrus aurantium L. fruit juice (CAde) on the regulation of 3T3-L1 cells adipocyte differentiation and function in vitro. We found that CAde enhances terminal adipocyte differentiation of 3T3-L1 cells raising the expression of CCAAT/enhancer binding protein beta (C/Ebpβ), peroxisome proliferator activated receptor gamma (Pparγ), glucose transporter type 4 (Glut4) and fatty acid binding protein 4 (Fabp4). CAde improves insulin-induced glucose uptake of 3T3-L1 adipocytes, as well. A focused analysis of the phases occurring in the pre-adipocytes differentiation to mature adipocytes furthermore revealed that CAde promotes the early differentiation stage by up-regulating C/ebpβ expression at 2, 4 and 8 h post the adipogenic induction and anticipating the 3T3-L1 cell cycle entry and progression during mitotic clonal expansion (MCE). These findings provide evidence that the exposure to CAde enhances in vitro fat cell differentiation of pre-adipocytes and functional capacity of mature adipocytes, and pave the way to the development of products derived from Citrus aurantium L. fruit juice, which may improve WAT functional capacity and may be effective for the prevention and/or treatment of T2D.

Background First-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR. Results Sequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM -siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM -siRNA significantly expanded hydrogen peroxide (H 2 O 2 )-induced senescence, while H 2 O 2 did not affect TFAM expression. Conclusions Histone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk. Graphical abstract

Adipose-derived stem cells (ADSCs) play a crucial role in angiogenesis and repair of damaged tissues. However, in pathological conditions including diabetes, ADSC function is compromised. This work aims at evaluating the effect of Methylglyoxal (MGO), a product of chronic hyperglycemia, on mouse ADSCs’ (mADSCs) pro-angiogenic function and the molecular mediators involved. The mADSCs were isolated from C57bl6 mice. MGO-adducts and p-p38 MAPK protein levels were evaluated by Western Blot. Human retinal endothelial cell (hREC) migration was analyzed by transwell assays. Gene expression was measured by qRT-PCR, and SA-βGal activity by cytofluorimetry. Soluble factor release was evaluated by multiplex assay. MGO treatment does not impair mADSC viability and induces MGO-adduct accumulation. hREC migration is reduced in response to both MGO-treated mADSCs and conditioned media from MGO-treated mADSCs, compared to untreated cells. This is associated with an increase of SA-βGal activity, SASP factor release and p53 and p21 expression, together with a VEGF- and PDGF-reduced release from MGO-treated mADSCs and a reduced p38-MAPK activation in hRECs. The MGO-induced impairment of mADSC function is reverted by senolytics. In conclusion, MGO impairs mADSCs’ pro-angiogenic function through the induction of a senescent phenotype, associated with the reduced secretion of growth factors crucial for hREC migration.

The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15) mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15)). In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15) cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15). These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1). Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4) restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15) by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia, responsible for the onset of several long-term complications. Recent evidence suggests that cognitive dysfunction represents an emerging complication of DM, but the underlying molecular mechanisms are still obscure. Dopamine (DA), a neurotransmitter essentially known for its relevance in the regulation of behavior and movement, modulates cognitive function, too. Interestingly, alterations of the dopaminergic system have been observed in DM. This review aims to offer a comprehensive overview of the most relevant experimental results assessing DA’s role in cognitive function, highlighting the presence of dopaminergic dysfunction in DM and supporting a role for glucotoxicity in DM-associated dopaminergic dysfunction and cognitive impairment. Several studies confirm a role for DA in cognition both in animal models and in humans. Similarly, significant alterations of the dopaminergic system have been observed in animal models of experimental diabetes and in diabetic patients, too. Evidence is accumulating that advanced glycation end products (AGEs) and their precursor methylglyoxal (MGO) are associated with cognitive impairment and alterations of the dopaminergic system. Further research is needed to clarify the molecular mechanisms linking DM-associated dopaminergic dysfunction and cognitive impairment and to assess the deleterious impact of glucotoxicity.