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OBJECTIVE--Previous studies show that polyunsaturated fatty acids (PUFAs) increase the insulin secretory capacity of pancreatic β-cells. We aimed at identifying PUFA-derived mediators and their cellular targets that are involved in the amplification of insulin release from β-cells preexposed to high glucose levels. RESEARCH DESIGN AND METHODS--The content of fatty acids in phospholipids of INS-1E β-cells was determined by lipidomics analysis. High-performance liquid chromatography was used to identify peroxidation products in β-cell cultures. Static and dynamic glucose-stimulated insulin secretion (GSIS) assays were performed on isolated rat islets and/or INS-1E cells. The function of peroxisome proliferator-activated receptor-δ (PPAR-δ) in regulating insulin secretion was investigated using pharmacological agents and gene expression manipulations. RESULTS--High glucose activated [cPLA.sub.2] and, subsequently, the hydrolysis of arachidonic and linoleic acid (AA and LA, respectively) from phospholipids in INS-1E cells. Glucose also increased the level of reactive oxygen species, which promoted the peroxidation of these PUFAs to generate 4-hydroxy-2E-nonenal (4-HNE). The latter mimicked the GSIS-amplffying effect of high glucose preexposure and of the PPAR-5 agonist GW501516 in INS-1E cells and isolated rat islets. These effects were blocked with GSK0660, a selective PPAR-8 antagonist, and the antioxidant N-acetylcysteine or by silencing PPAR-δ expression. High glucose, 4-HNE, and GW501516 also induced luciferase expression in a PPAR-δ-mediated transactivation assay. Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range. CONCLUSIONS--Elevated glucose levels augment the release of AA and LA from phospholipids and their peroxidation to 4-HNE in β-cells. This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells. Diabetes 60:2830-2842, 2011, The effect of glucose on β-cell function depends on its concentration and duration of exposure (1). An acute exposure to high glucose triggers the classical biphasic insulin secretion as a [...]

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 12-1ipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-1ipoxygenase, 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., Hyperglycemia is a major and independent risk factor in the development of cardiovascular disease and atherosclerosis in diabetes (1,2). Vascular endothelial cell (VEC) dysfunction precedes the development of atherosclerotic plaques [...]

Membrane glycoprotein PC-1, an inhibitor of insulin signaling, produces insulin resistance when overexpressed in cells transfected with PC-1 cDNA. In the present study, we determined whether PC-l plays a role [...]

Hyperglycemia has been implicated in the pathogenesis of both micro- and macrovascular complications in diabetes. Little is known, however, about glucose transporters and their regulation in the vascular system. In [...]

Enhanced production of arachidonic acid metabolites of the lipoxygenase (LOX) pathway, i.e. hydroxyeicosatetraenoic acids (HETEs), may be linked to diabetes-related complications. Here we studied the effects of the LOX inhibitor [...]

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of pathologies, ranging from hepatocellular steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. It has been suggested that fish oil containing n-3 polyunsaturated fatty acids (n-3 PUFA) induce beneficial effects in NAFLD. However, n-3 PUFA are sensitive to peroxidation that generate free radicals and reactive aldehydes. We aimed at determining whether changing the tissue ratio of n-3 to n-6 PUFA may be beneficial or alternatively harmful to the etiology of NAFLD. The transgenic Fat-1mouse model was used to determine whether n-3 PUFA positively or negatively affect the development of NAFLD. fat-1mice express the fat-1gene of Caenorhabditis elegans, which encodes an n-3 fatty-acid desaturase that converts n-6 to n-3 fatty acids. Wild-type C57BL/6 mice served as the control group. Both groups of mice were fed methionine and choline deficient (MCD) diet, which induces NASH within 4 weeks. The study shows that NASH developed faster and was more severe in mice from the fat-1 group when compared to control C57BL/6 mice. This was due to enhanced lipid peroxidation of PUFA in the liver of the fat-1mice as compared to the control group. Results of our mice study suggest that supplementing the diet of individuals who develop or have fatty livers with n-3 PUFA should be carefully considered and if recommended adequate antioxidants should be added to the diet in order to reduce such risk.

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.

Intact actin microfilaments are necessary for insulin-regulated GLUT4 translocation from intracellular pools to the plasma membrane. Products of the lipoxygenase (LO) pathway were shown to be implicated in the regulation of actin cytoskeleton rearrangement. The aim of this study was to examine the role of these LO products for cardiac insulin signaling and glucose uptake, GLUT4 translocation, and actin-based cytoskeleton structure. Exposure of cardiomyocytes to esculetin or NDGA, two structurally different LO inhibitors, induced a complete inhibition of insulin-stimulated glucose uptake, whereas control cells showed a threefold stimulation by insulin. Addition of 12(S)-HETE rendered the NDGA-treated cells insulin-sensitive. Early insulin signaling was not changed in cells exposed to LO inhibitors. Cell surface biotinylation of control cells showed a twofold increase of GLUT4 at the cell surface after insulin stimulation. In contrast, the LO inhibitors induced a complete inhibition of insulin-stimulated GLUT4 translocation. Labeling of the F-actin cytoskeleton revealed a prominent disassembly of actin fibers in cells exposed to the LO inhibitors. In conclusion, we show here that products of the LO reaction participate in the organization of the actin network in ventricular cardiomyocytes. Inhibition of LO blocks GLUT4 translocation without affecting insulin signaling events. These data suggest that products of the LO reaction participate in the regulation of glucose transport by contribution to a rearrangement of actin cytoskeletal elements.

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 F2α, 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.

Intact actin microfilaments are required for insulin-regulated glucose transporter isoform 4 (GLUT4) translocation to the plasma membrane. Lipoxygenase (LO) metabolites have recently been shown to contribute to the regulation of actin cytoskeleton rearrangement. In the present investigation, ventricular cardiomyocytes were used to study the effects of two structurally different LO inhibitors (esculetin and nordihydroguaiaretic acid) on insulin signalling events, glucose uptake, GLUT4 translocation and the actin network organization. Insulin stimulation increased glucose uptake 3-fold in control cells, whereas LO inhibition completely blocked this effect. This was paralleled by a slight reduction in the insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2. However, inhibition of 12-LO did not affect the association of phosphatidylinositol 3-kinase with IRS-1 and the phosphorylation of Akt/protein kinase B in response to insulin. Addition of 12(S)-hydroxyeicosatetraenoic acid almost completely restored the insulin action in cells exposed to nordihydroguaiaretic acid. Insulin stimulation increased cell surface GLUT4 2-fold in control cells, whereas LO inhibition abrogated the insulin-stimulated GLUT4 translocation. LO inhibition induced a prominent disassembly of actin fibres compared with control cells. In conclusion, we show here that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of the actin network in cardiomyocytes. LO inhibition blocks GLUT4 translocation without affecting downstream insulin signalling. These data suggest that LO metabolites participate in the regulation of glucose transport by contributing to a rearrangement of actin cytoskeletal elements.

T helper cell (Th)1-primed CD4 T cells from wild-type donors make little interleukin (IL)-4 when restimulated under Th2 conditions. However, such restimulation of Th1-primed cells from interferon (IFN)-γ2/− or IFN-γ receptor (IFN-γR)−/− mice resulted in substantial production of IL-4 and other Th2 cytokines. Adding IFN-γ to the priming culture markedly diminished the capacity of Th1-primed IFN-γ2/− cells to express IL-4. Even IFN-γ–producing cells from IFN-γR−/− mice could acquire IL-4–producing capacity. Thus, IFN-γ is not required for the development of IFN-γ–producing capacity, but it plays a critical role in suppressing the IL-4–producing potential of Th1 cells.

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4 – 8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10 – 16 h. In the present study, we employed the ‘membrane sheet assay’ in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the ‘sheet assay’ revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2=18 min) than that of glucose deprivation (T1/2>60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.

Naive CD4⁺ T cells differ from memory cells by their heightened expression of the disialoceramide recognized by antibody 3G11. 3G11^bright cells respond well to immobilized anti-CD3 / anti-CD28 and to their cognate antigens but produce little or no IFN-γ or IL-4 "acutely" and undergo cell death even in the presence of IL-2. They can be rescued by IL-4, IL-6 or IL-12. IL-6 is particularly notable since it is neutral in regard to Th1 / Th2 priming, allowing an assessment of the role of endogenous IL-4 in priming for IL-4 production. Naive TCR-transgenic BALB / c scid T cells cultured with an ovalbumin peptide and IL-4^– / – antigen-presenting cells in the presence of IL-6 showed a modest degree of priming for IL-4 production if both IFN-γ and IL-12 were neutralized. This priming is far less than that observed if IL-4 is added to the priming culture. These results indicate that IL-4 production as a result of TCR engagement is sufficient for only a minor component of the polarization observed when unseparated BALB / c CD4 T cell populations are primed or when IL-4 is intentionally added to the priming culture.

In order to determine the possible role of DNA methylation as a regulatory mechanism for the restricted pattern of lymphokine production among differentiated Th1and Th2cells, we examined the extent of methylation of the interferon γ (IFN-γ) and the interleukin 4 (IL-4) genes in fresh activated murine Th0, Th1and Th2cells, unstimulated naive T cells, B cells, bone marrow derived non-B non-T cells, thymocytes and liver. All of the CpG dinucleotides examined in the IL-4 and the IFN-γ genes, were fully methylated over the body of the gene in all of the examined cells. However, analysis of the promoter regions of these genes revealed a different pattern. While the IL-4 promoter is fully methylated in all of the examined cells, two adjacent CpG dinucleotides near the initiation point of the IFN-γ gene were unmethylated in all T cells, including 17-day-old fetal thymocytes. In contrast, B cells, bone marrow non-B non-T cells and liver cells displayed a full methylated profile of the IFNγ promoter. These results suggest that the mutually exclusive pattern of IFNγ and IL-4 production in Th1and Th2cells is not regulated by differential demethylation of these two genes.

Primary cultures of skeletal muscle cells secrete plasminogen activator (PA) activity to the conditioning medium and display membrane-bound PA. Growth of these cells in culture in presence of 10-7 M dexamethasone resulted in a marked reduction of the membranal and secreted PA activity. The hormone also reduced cytosolic creative phosphokinase (CPK) activity and cytosolic protein content. However, cell viability and their ability to undergo fusion were uneffected. The extent of hormone-induced reduction in PA activity depended on the time and extend of exposure. Maximal suppression was obtained by exposing the cells to dexamethasone during the first 4 days of culture. The medium conditioned with dexamethasone-treated cells did not inhibit plasmin, endogenous PA or exogenous PA. Exposure of the conditioned medium from hormone-treated cells to sodium dodecyl sulphate (SDS) or trypsin restored the activity to values observed in media from cells not exposed to the hormone. Acidification of the medium failed to reactivate the enzyme. The myogenic cell line L-8 also displayed membrane-bound PA activity, which was of a comparable magnitude in both fusing and non-fusing L-8 cells. However, in contrast to the primary cultures, exposure of L-8 cells to dexamethasone had no effect on their PA activity whether studied under conditions which allowed or prohibited fusion. The present findings imply that PA has no conducive role in the process of fusion associated with maturation of skeletal muscle cells.

This study examines the mechanisms of glucocorticoid-induced insulin resistance in rat soleus muscle. Glucocorticoid excess was induced by administration of dexamethasone to rats for 5 days. Dexamethasone decreased the sensitivity of 3-O-methylglucose transport, 2-deoxyglucose phosphorylation, glycogen synthesis and glucose oxidation to insulin. The total content of GLUT4 glucose transporters was not decreased by dexamethasone; however, the increase in these transporters in the plasma membrane in response to insulin (100 m-units/litre) was lessened. In contrast, the sensitivity of lactate formation to insulin was normal. The content of 2-deoxyglucose in the dexamethasone-treated muscle was decreased at 100 m-units/litre insulin, while the contents of glucose 6-phosphate and fructose 2,6-bisphosphate were normal at all concentrations of insulin studied. The maximal activity of hexokinase in the soleus muscle was not affected by dexamethasone; however, inhibition of this enzyme by glucose 6-phosphate was decreased. These results suggest the following. (1) Glucocorticoid excess causes insulin resistance in skeletal muscle by directly inhibiting the translocation of the GLUT4 glucose transporters to the plasma membrane in response to insulin; since the activity of hexokinase is not affected, the changes in the sensitivity of glucose phosphorylation to insulin seen under these conditions are secondary to those in glucose transport. (2) The sensitivity of glycogen synthesis and glucose oxidation to insulin is decreased, but that of glycolysis is not affected: a redistribution of glucose away from the pathway of glycogen synthesis and glucose oxidation could maintain a normal rate of lactate formation although the rate of glucose transport is decreased.

We have previously demonstrated that alloantisera prepared by reciprocal immunization of strain 2 and strain 13 guinea pigs specifically block stimulation of in vitro DNA synthesis in genetically controlled systems. In order to determine whether this blockade extends to other T-lymphocyte functions, we examined the effect of alloantisera on the production of migration inhibition factor (MIF). (2 x 13)F1 guinea pigs were immunized with a DNP derivative of the copolymer of L-glutamic acid and L-lysine (DNP-GL) and with DNP guinea pig albumin (GPA). The response to the former is controlled by a 2-linked Ir gene while that to the latter is mainly controlled by a 13-linked Ir gene. MIF production was assayed by an indirect procedure in which the migrating cell population lacked the histocompatibility antigen against which the alloantiserum was directed. Our results showed that anti-2 serum blocked MIF production by F1 cells in response to DNP-GL but not DNP-GPA while anti-13 serum had the opposite effect. These experiments show that expression of a second major T-cell function is specifically blocked by alloantisera and suggest that Ir-gene products may act as antigen recognition substances on more than one type of T cell.

Insulin secretion from pancreatic beta cells is tightly regulated by glucose and paracrine signals within the microenvironment of islets of Langerhans. Extracellular matrix from islet microcapillary endothelial cells (IMEC) affect beta-cell spreading and amplify insulin secretion. This study was aimed at investigating the hypothesis that contact-independent paracrine signals generated from IMEC may also modulate beta-cell insulin secretory functions. For this purpose, conditioned medium (CMp) preparations were prepared from primary cultures of rat IMEC and were used to simulate contact-independent beta cell–endothelial cell communication. Glucose-stimulated insulin secretion (GSIS) assays were then performed on freshly isolated rat islets and the INS-1E insulinoma cell line, followed by fractionation of the CMp, mass spectroscopic identification of the factor, and characterization of the mechanism of action. The IMEC-derived CMp markedly attenuated first- and second-phase GSIS in a time- and dose-dependent manner without altering cellular insulin content and cell viability. Size exclusion fractionation, chromatographic and mass-spectroscopic analyses of the CMp identified the attenuating factor as the enzyme triosephosphate isomerase (TPI). An antibody against TPI abrogated the attenuating activity of the CMp while recombinant human TPI (hTPI) attenuated GSIS from beta cells. This effect was reversed in the presence of tolbutamide in the GSIS assay. In silico docking simulation identified regions on the TPI dimer that were important for potential interactions with the extracellular epitopes of the sulfonylurea receptor in the complex. This study supports the hypothesis that an effective paracrine interaction exists between IMEC and beta cells and modulates glucose-induced insulin secretion via TPI–sulfonylurea receptor–KATPchannel (SUR1-Kir6.2) complex attenuating interactions.