Evgenia Alpert

A natural protective mechanism against hyperglycaemia in vascular endothelial and smooth-muscle cells: role of glucose and 12-hydroxyeicosatetraenoic acid

Bovine aortic endothelial and smooth-muscle cells down-regulate the rate of glucose transport in the face of hyperglycaemia, thus providing protection against deleterious effects of increased intracellular glucose levels. When exposed to high glucose concentrations these cells reduced the mRNA and protein content of their typical glucose transporter, GLUT-1, as well as its plasma-membrane abundance. Inhibition of the lipoxygenase (LO) pathway, and particularly 12-LO, reversed this glucose-induced down-regulatory process and restored the rate of hexose transport to the level seen in vascular cells exposed to normal glucose levels. This reversal was accompanied by increased levels of GLUT-1 mRNA and protein, as well as of its plasma-membrane content. Exposure of the vascular cells to elevated glucose concentrations increased by 2-3-fold the levels of cell-associated and secreted 12-hydroxyeicosatetraenoic acid (12-HETE), the product of 12-LO. Inhibition of 15- and 5-LO, cyclo-oxygenases 1 and 2, and eicosanoid-producing cytochrome P450 did not modify the hexose-transport system in vascular cells. These results suggest a role for HETEs in the autoregulation of hexose transport in vascular cells. 8-Iso prostaglandin F(2alpha), a non-enzymic oxidation product of arachidonic acid, had no effect on the hexose-transport system in vascular cells exposed to hyperglycaemic conditions. Taken together, these findings show that hyperglycaemia increases the production rate of 12-HETE, which in turn mediates the down-regulation of GLUT-1 expression and the glucose-transport system in vascular endothelial and smooth-muscle cells.

The roles of hyperglycaemia and oxidative stress in the rise and collapse of the natural protective mechanism against vascular endothelial cell dysfunction in diabetes

Abstract Vascular endothelial cell (VEC) dysfunction in diabetes has been associated with hyperglycaemia-induced intra- and extracellular glycation of proteins and to overproduction of glucose-derived free radicals. VEC protect their intracellular environment against an increased influx of glucose in face of hyperglycaemia by reducing the expression and plasma membrane abundance of their glucose transporter-1 (GLUT-1). We investigated the hypothesis that glucose-derived free radicals induce this down-regulatory mechanism in VEC, but proved the contrary. In fact, pro-oxidants significantly increased the expression and plasma membrane abundance of GLUT-1 and the rate of glucose transport in VEC while abolishing high-glucose-induced down-regulation of the hexose transport system. The resulting uncontrolled influx of glucose followed by overproduction of glucose-derived ROS further up-regulates the rate of glucose transport, and vice versa. This perpetuating glycoxidative stress finally leads to the collapse of the auto-regulatory protective mechanism and accelerates the development of dysfunctional endothelium in blood vessels.

The Natural Protective Mechanism Against Hyperglycemia in Vascular Endothelial Cells Roles of the Lipid Peroxidation Product 4-Hydroxydodecadienal and Peroxisome Proliferator–Activated Receptor δ

OBJECTIVE Vascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid–metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator–activated receptor (PPAR) δ was investigated. RESEARCH DESIGN AND METHODS Effects of 4-HDDE and PPARδ on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions. RESULTS Using GW501516 (PPARδ agonist) and GSK0660 (PPARδ antagonist), we discovered that high-glucose–induced downregulation of the glucose transport system in VECs is mediated by PPARδ. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARδ in cells overexpressing human PPAR (hPPAR)δ but not hPPARα, -γ1, or -γ2. Moreover, silencing of PPARδ prevented high-glucose–dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARδ to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay. CONCLUSIONS Collectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARδ.

The Natural Protective Mechanism Against Hyperglycemia in Vascular Endothelial Cells: Roles of the Lipid Peroxidation Product 4-Hydroxydodecadienal (4-HDDE) and PPARδ

OBJECTIVE Vascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid–metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator–activated receptor (PPAR) δ was investigated. RESEARCH DESIGN AND METHODS Effects of 4-HDDE and PPARδ on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions. RESULTS Using GW501516 (PPARδ agonist) and GSK0660 (PPARδ antagonist), we discovered that high-glucose–induced downregulation of the glucose transport system in VECs is mediated by PPARδ. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARδ in cells overexpressing human PPAR (hPPAR)δ but not hPPARα, -γ1, or -γ2. Moreover, silencing of PPARδ prevented high-glucose–dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARδ to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay. CONCLUSIONS Collectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARδ.

Delayed autoregulation of glucose transport in vascular endothelial cells

Aims/hypothesisWe aimed to characterise the development of autoregulation of glucose transport in vascular endothelial cells and its relationship to 12-lipoxygenase (12-LO) expression.MethodsBovine aortic endothelial cells were exposed to 5.5 and 23.0xa0mmol/l glucose for up to 48xa0h. The rates of glucose transport, GLUT-1 and 12-LO expression and of 12-hydroxyeicosatetraenoic acid (12-HETE) production were determined.ResultsWe showed high glucose-dependent downregulation of glucose transport and transporter in vascular endothelial cells within 36–48xa0h. A similar time-dependent increase in the expression of 12-LO and the generation of its product 12-HETE was also observed. This downregulatory process was prevented when lipoxygenase activity was inhibited.Conclusions/interpretationVascular endothelial cells, which were previously thought to be “glucose-blind”, do in fact downregulate GLUT-1 expression and the rate of glucose transport in response to extended exposure to high glucose concentrations. This slow development of glucose-induced downregulation in vascular endothelial cells is related to the slower basal rate of glucose transport in these cells and the slow induction of 12-LO. These data are interesting in view of current hypotheses that attribute vascular endothelial cell dysfunction in diabetes to the lack of a glucose-induced autoregulatory response.

Novel D-xylose derivatives stimulate muscle glucose uptake by activating AMP-activated protein kinase alpha.

Type 2 diabetes mellitus has reached epidemic proportions; therefore, the search for novel antihyperglycemic drugs is intense. We have discovered that D-xylose increases the rate of glucose transport in a non-insulin-dependent manner in rat and human myotubes in vitro. Due to the unfavorable pharmacokinetic properties of D-xylose we aimed at synthesizing active derivatives with improved parameters. Quantitative structure-activity relationship analysis identified critical hydroxyl groups in D-xylose. These data were used to synthesize various hydrophobic derivatives of D-xylose of which compound 19 the was most potent compound in stimulating the rate of hexose transport by increasing the abundance of glucose transporter-4 in the plasma membrane of myotubes. This effect resulted from the activation of AMP-activated protein kinase without recruiting the insulin transduction mechanism. These results show that lipophilic D-xylose derivatives may serve as prototype molecules for the development of novel antihyperglycemic drugs for the treatment of diabetes.

Cyclooxygenase-2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in an insulin- and AMPKα-independent manner

Aims/hypothesisSome cyclooxygenase-2 (COX2, also known as prostaglandin-endoperoxide synthase 2 [PTGS2]) inhibitors have been shown to increase insulin sensitivity in man or induce hypoglycaemic episodes when overconsumed or taken in combination with oral hypoglycaemic drugs. These side-effects and their impact on patients are not always recognised in routine clinical practice. We investigated whether these side-effects of COX2 (PTGS2) inhibitors result from stimulation of the glucose transport system in skeletal muscle cells.Materials and methodsL6 myotube cultures were used to study effects of COX2 (PTGS2) inhibitors on the glucose transport system and their relationship to PTGS2 expression, insulin action and AMP-activated protein kinase α (AMPKα) activity.ResultsThe inhibitors niflumic acid, nimesulide and rofecoxib increased the rate of hexose uptake in L6 myotubes in the absence of insulin and in a dose- and time-dependent manner. They did this by increasing the total cell content of member 4 of the solute carrier family 2 (SCLC2A4, previously known as glucose transporter 4 [GLUT4]) (but not SCLC2A1 [previously known as GLUT1]) mRNA and protein and the amount of it in the plasma membrane. AMPKα was not involved in the latter effect since the inhibitors did not activate it. In addition, none of the inhibitors modulated the rate of hexose transport in vascular endothelial and smooth muscle cells expressing PTGS2 and SCLC2A1. Prostaglandin-endoperoxide synthase 1 (also known as cyclooxygenase 1) inhibitors (acetylsalicylic acid and indomethacin) did not alter the rate of hexose uptake and SCLC2A4 subcellular distribution in L6 myotubes.Conclusions/interpretationThis study suggests that certain COX2 (PTGS2) inhibitors can alter glucose homeostasis in vivo by stimulating glucose uptake in skeletal muscles that express PTGS2.

Beta Cells Secrete Significant and Regulated Levels of Insulin for Long Periods when Seeded onto Acellular Micro-Scaffolds.

The aim of this work is to obtain significant and regulated insulin secretion from human beta cells ex vivo. Long-term culture of human pancreatic islets and attempts at expanding human islet cells normally result in loss of beta-cell phenotype. We propose that to obtain proper ex vivo beta cell function, there is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. We here describe the preparation of endocrine micro-pancreata (EMPs) that are made up of acellular organ-derived micro-scaffolds seeded with human intact or enzymatically dissociated islets. We show that EMPs constructed by seeding whole islets, freshly enzymatically-dissociated islets or even dissociated islets grown first in standard monolayer cultures express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than 3 months in vitro.