Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

The Combination of Whole Cell Lipidomics Analysis and Single Cell Confocal Imaging of Fluidity and Micropolarity Provides Insight into Stress-Induced Lipid Turnover in Subcellular Organelles of Pancreatic Beta Cells.

Modern omics techniques reveal molecular structures and cellular networks of tissues and cells in unprecedented detail. Recent advances in single cell analysis have further revolutionized all disciplines in cellular and molecular biology. These methods have also been employed in current investigations on the structure and function of insulin secreting beta cells under normal and pathological conditions that lead to an impaired glucose tolerance and type 2 diabetes. Proteomic and transcriptomic analyses have pointed to significant alterations in protein expression and function in beta cells exposed to diabetes like conditions (e.g., high glucose and/or saturated fatty acids levels). These nutritional overload stressful conditions are often defined as glucolipotoxic due to the progressive damage they cause to the cells. Our recent studies on the rat insulinoma-derived INS-1E beta cell line point to differential effects of such conditions in the phospholipid bilayers in beta cells. This review focuses on confocal microscopy-based detection of these profound alterations in the plasma membrane and membranes of insulin granules and lipid droplets in single beta cells under such nutritional load conditions.

Regulation of GLUT4 activity in myotubes by 3-O-methyl-d-glucose.

The rate of glucose influx to skeletal muscles is determined primarily by the number of functional units of glucose transporter-4 (GLUT4) in the myotube plasma membrane. The abundance of GLUT4 in the plasma membrane is tightly regulated by insulin or contractile activity, which employ distinct pathways to translocate GLUT4-rich vesicles from intracellular compartments. Various studies have indicated that GLUT4 intrinsic activity is also regulated by conformational changes and/or interactions with membrane components and intracellular proteins in the vicinity of the plasma membrane. Here we show that the non-metabolizable glucose analog 3-O-methyl-d-glucose (MeGlc) augmented the rate of hexose transport into myotubes by increasing GLUT4 intrinsic activity without altering the content of the transporter in the plasma membrane. This effect was not a consequence of ATP depletion or hyperosmolar stress and did not involve Akt/PKB or AMPK signal transduction pathways. MeGlc reduced the inhibitory potency (increased Ki) of indinavir, a selective inhibitor of GLUT4, in a dose-dependent manner. Kinetic analyses indicate that MeGlc induced changes in GLUT4 or GLUT4 complexes within the plasma membrane, which enhanced the hexose transport activity and reduced the potency of indinavir inhibition. Finally, we present a simple kinetic analysis for screening and discovering low molecular weight compounds that augment GLUT4 activity.

Deficiency in apolipoprotein A-I ablates the pharmacological effects of metformin on plasma glucose homeostasis and hepatic lipid deposition.

Recently, we showed that deficiency in apolipoprotein A-I (ApoA-I) sensitizes mice to diet-induced obesity, glucose intolerance and NAFLD. Here we investigated the potential involvement of ApoA-I in the pharmacological effects of metformin on glucose intolerance and NAFLD development. Groups of apoa1-deficient (apoa1(-/-)) and C57BL/6 mice fed western-type diet were either treated with a daily dose of 300 mg/kg metformin for 18 weeks or left untreated for the same period. Then, histological and biochemical analyses were performed. Metformin treatment led to a comparable reduction in plasma insulin levels in both C57BL/6 and apoa1(-/-) mice following intraperitoneal glucose tolerance test. However, only metformin-treated C57BL/6 mice maintained sufficient peripheral insulin sensitivity to effectively clear glucose following the challenge, as indicated by a [(3)H]-2-deoxy-D-glucose uptake assay in isolated soleus muscle. Similarly, deficiency in ApoA-I ablated the effect of metformin on hepatic lipid deposition and NAFLD development. Gene expression analysis indicated that the effects of ApoA-I on metformin treatment may be independent of adenosine monophosphate-activated protein kinase (AMPK) activation and de novo lipogenesis. Interestingly, metformin treatment reduced mitochondrial oxidative phosphorylation function only in apoa1(-/-) mice. Our data show that the role of ApoA-I in diabetes extends to the modulation of the pharmacological actions of metformin, a common drug for the treatment of type 2 diabetes.

Regulation of Nox enzymes expression in vascular pathophysiology: Focusing on transcription factors and epigenetic mechanisms.

NADPH oxidases (Nox) represent a family of hetero-oligomeric enzymes whose exclusive biological function is the generation of reactive oxygen species (ROS). Nox-derived ROS are essential modulators of signal transduction pathways that control key physiological activities such as cell growth, proliferation, migration, differentiation, and apoptosis, immune responses, and biochemical pathways. Enhanced formation of Nox-derived ROS, which is generally associated with the up-regulation of different Nox subtypes, has been established in various pathologies, namely cardiovascular diseases, diabetes, obesity, cancer, and neurodegeneration. The detrimental effects of Nox-derived ROS are related to alterations in cell signalling and/or direct irreversible oxidative damage of nucleic acids, proteins, carbohydrates, and lipids. Thus, understanding of transcriptional regulation mechanisms of Nox enzymes have been extensively investigated in an attempt to find ways to counteract the excessive formation of Nox-derived ROS in various pathological states. Despite the numerous existing data, the molecular pathways responsible for Nox up-regulation are not completely understood. This review article summarizes some of the recent advances and concepts related to the regulation of Nox expression in the vascular pathophysiology. It highlights the role of transcription factors and epigenetic mechanisms in this process. Identification of the signalling molecules involved in Nox up-regulation, which is associated with the onset and development of cardiovascular dysfunction may contribute to the development of novel strategies for the treatment of cardiovascular diseases.

Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner.

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated ß-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.

High-glucose-increased expression and activation of NADPH oxidase in human vascular smooth muscle cells is mediated by 4-hydroxynonenal-activated PPARα and PPARß/δ.

High glucose induces vascular smooth muscle cell (SMC) dysfunction by generating oxidative stress attributable, in part, to the up-regulated NADPH oxidases (Nox). We have attempted to elucidate the high-glucose-generated molecular signals that mediate this effect and hypothesize that products of high-glucose-induced lipid peroxidation regulate Nox by activating peroxisome proliferator-activated receptors (PPARs). Human aortic SMCs were exposed to glucose (5.5-25 mM) or 4-hydroxynonenal (1-25 µM, 4-HNE). Lucigenin assay, real-time polymerase chain reaction, western blot, and promoter analyses were employed to investigate Nox. We found that high glucose generated an increase in Nox activity and expression. It also promoted oxidative stress that consequently induced lipid peroxidation, which resulted in the production of 4-HNE. Pharmacological inhibition of Nox activity significantly reduced the formation of high-glucose-induced 4-HNE. Exposure of SMCs to non-cytotoxic concentrations (1-10 µM) of 4-HNE alone mimicked the effect of high glucose incubation, whereas scavenging of 4-HNE by N-acetyl L-cysteine completely abolished both the effects of high glucose and 4-HNE. The latter exerted its effect by activating PPARα and PPARß/δ, but not PPARγ, as assessed pharmacologically by the inhibitory effect of selective antagonists and following the silencing of the expression of these receptors. These new data indicate that 4-HNE, generated following Nox activation, functions as an endogenous activator of PPARα and PPARß/δ. The newly discovered "lipid peroxidation products-PPARs-Nox axis" represents a novel mechanism of Nox regulation and an additional therapeutic target for oxidative stress in diabetes.

Regulatory T-cells and cAMP suppress effector T-cells independently of PKA-CREM/ICER: a potential role for Epac.

cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.

Synthesis and mechanism of hypoglycemic activity of benzothiazole derivatives.

Adenosine 5'-monophosphate activated protein kinase (AMPK) has emerged as a major potential target for novel antidiabetic drugs. We studied the structure of 2-chloro-5-((Z)-((E)-5-((5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-4-oxothiazolidin-2-ylidene)amino)benzoic acid (PT-1), which attenuates the autoinhibition of the enzyme AMPK, for the design and synthesis of different benzothiazoles with potential antidiabetic activity. We synthesized several structurally related benzothiazole derivatives that increased the rate of glucose uptake in L6 myotubes in an AMPK-dependent manner. One compound, 2-(benzo[d]thiazol-2-ylmethylthio)-6-ethoxybenzo[d]thiazole (34), augmented the rate of glucose uptake up to 2.5-fold compared with vehicle-treated cells and up to 1.1-fold compared to PT-1. Concomitantly, it elevated the abundance of GLUT4 in the plasma membrane of the myotubes and activated AMPK. Subcutaneous administration of 34 to hyperglycemic Kuo Kondo rats carrying the Ay-yellow obese gene (KKAy) mice lowered blood glucose levels toward the normoglycemic range. In accord with its activity, compound 34 showed a high fit value to a pharmacophore model derived from the PT-1.

Oleic acid and adipokines synergize in inducing proliferation and inflammatory signalling in human vascular smooth muscle cells.

In the context of obesity, perivascular fat produces various adipokines and releases free fatty acids, which may induce inflammation and proliferation in the vascular wall. In this study we investigated how adipokines, oleic acid (OA) and the combined treatment regulate human vascular smooth muscle cell (hVSMC) proliferation and migration and the underlying signalling pathways. Adipocyte-conditioned media (CM) generated from human adipocytes induces a prominent proliferation and migration of hVSMC. Autocrine action of adiponectin totally abolishes CM-induced proliferation. Furthermore, OA but not palmitic acid induces proliferation of hVSMC. CM itself does not contain fatty acids, but CM in combination with OA markedly enhances proliferation of hVSMC in a synergistic way. Both the nuclear factor (NF)-κB and the mammalian target of rapamycin (mTOR) pathway were synergistically activated under these conditions and found to be essential for hVSMC proliferation. Expression of iNOS and production of nitric oxide was only enhanced by combined treatment inducing a marked release of VEGF. Combination of OA and VEGF induces an additive increase of hVSMC proliferation. We could show that the combination of CM and OA led to a synergistic proliferation of hVSMC. Expression of iNOS and production of nitric oxide were only enhanced under these conditions and were paralleled by a marked release of VEGF. These results suggest that the combined elevated release of fatty acids and adipokines by adipose tissue in obesity might be critically related to hVSMC dysfunction, vascular inflammation and the development of atherosclerosis.

Pathological aspects of lipid peroxidation.

Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases. The current evidence for the implication of LPO in pathological processes is discussed in this review. New data and literature review are provided evaluating the role of LPO in the pathophysiology of ageing and classically oxidative stress-linked diseases, such as neurodegenerative diseases, diabetes and atherosclerosis (the main cause of cardiovascular complications). Striking evidences implicating LPO in foetal vascular dysfunction occurring in pre-eclampsia, in renal and liver diseases, as well as their role as cause and consequence to cancer development are addressed.

PDE8 regulates rapid Teff cell adhesion and proliferation independent of ICER.

BACKGROUND: Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. METHODOLOGY/PRINCIPAL FINDINGS: We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem-/- mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. CONCLUSION/SIGNIFICANCE: Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

Adenosine Monophosphate-Activated Protein Kinase (AMPK) as a New Target for Antidiabetic Drugs: A Review on Metabolic, Pharmacological and Chemical Considerations.

In view of the epidemic nature of type 2 diabetes and the substantial rate of failure of current oral antidiabetic drugs the quest for new therapeutics is intensive. The adenosine monophosphate-activated protein kinase (AMPK) is an important regulatory protein for cellular energy balance and is considered a master switch of glucose and lipid metabolism in various organs, especially in skeletal muscle and liver. In skeletal muscles, AMPK stimulates glucose transport and fatty acid oxidation. In the liver, it augments fatty acid oxidation and decreases glucose output, cholesterol and triglyceride synthesis. These metabolic effects induced by AMPK are associated with lowering blood glucose levels in hyperglycemic individuals. Two classes of oral antihyperglycemic drugs (biguanidines and thiazolidinediones) have been shown to exert some of their therapeutic effects by directly or indirectly activating AMPK. However, side effects and an acquired resistance to these drugs emphasize the need for the development of novel and efficacious AMPK activators. We have recently discovered a new class of hydrophobic D-xylose derivatives that activates AMPK in skeletal muscles in a non insulin-dependent manner. One of these derivatives (2,4;3,5-dibenzylidene-D-xylose-diethyl-dithioacetal) stimulates the rate of hexose transport in skeletal muscle cells by increasing the abundance of glucose transporter-4 (GLUT-4) in the plasma membrane through activation of AMPK. This compound reduces blood glucose levels in diabetic mice and therefore offers a novel strategy of therapeutic intervention strategy in type 2 diabetes. The present review describes various classes of chemically-related compounds that activate AMPK by direct or indirect interactions and discusses their potential for candidate antihyperglycemic drug development.

IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation.

IL-1 causes a marked increase in the degree of expansion of naïve and memory CD4 T cells in response to challenge with their cognate antigen. The response occurs when only specific CD4 T cells can respond to IL-1beta, is not induced by a series of other cytokines and does not depend on IL-6 or CD-28. When WT cells are primed in IL-1R1(-/-) recipients, IL-1 increases the proportion of cytokine-producing transgenic CD4 T cells, especially IL-17- and IL-4-producing cells, strikingly increases serum IgE levels and serum IgG1 levels. IL-1beta enhances antigen-mediated expansion of in vitro primed Th1, Th2, and Th17 cells transferred to IL-1R1(-/-) recipients. The IL-1 receptor antagonist diminished responses to antigen plus lipopolysaccharide (LPS) by approximately 55%. These results indicate that IL-1beta signaling in T cells markedly induces robust and durable primary and secondary CD4 responses.

Selective cyclooxygenase-2 inhibitors stimulate glucose transport in L6 myotubes in a protein kinase Cdelta-dependent manner.

Selective inhibitors of cyclooxygenase-2 (prostaglandin-endoperoxide synthase-2; COX-2) augment the rate of hexose uptake in myotubes by recruiting glucose transporter-4 (GLUT-4) to the plasma membrane in an insulin- and AMPKalpha-independent manner [Alpert E, Gruzman A, Lardi-Studler B, Cohen G, Reich R, Sasson S. Cyclooxygenase-2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in an insulin- and AMPKalpha-independent manner. Diabetologia 2006;49:562-70]. We aimed at elucidating the molecular interactions that mediate this effect of COX-2 inhibitors in L6 myotubes. The effects of the inhibitors niflumic acid, nimesulide and rofecoxib on activities and phosphorylation state of key proteins in the insulin transduction pathway were determined. These inhibitors did not induce specific tyrosine phosphorylation in IRS-1, could not assemble a functional IRS-PI3K-PKB/Akt complex and did not activate GSK3alpha/beta, JNK1/2, ERK1/2, p38-MAPK or c-Cbl by site-specific phosphorylation(s). Yet, like insulin, they activated mTOR and induced downstream threonine phosphorylation in p70S6K and 4EBP1. However, rapamycin, which inhibits mTOR enzymatic activity, did not interfere with COX-2 inhibitor-induced stimulation of hexose uptake in myotube. Thus, mTOR activation was not required for COX-2 inhibitor-dependent augmentation of hexose transport in myotubes. Because PKCdelta has also been shown to activate mTOR, we asked whether COX-2 inhibitors activate mTOR by a prior activation of PKCdelta. Indeed, all three inhibitors induced tyrosine phosphorylation in PKCdelta and stimulated its kinase activity. Moreover, pharmacological inhibition of PKCdelta or the expression of a dominant-negative form of PKCdelta in myotubes completely abolished COX-2 inhibitor-dependent stimulation of hexose uptake. This study shows that selective COX-2 inhibitors activate a unique PKCdelta-dependent pathway to increase GLUT-4 abundance in the plasma membrane of myotubes and augment the rate of hexose transport.

4-Hydroxy tempol-induced impairment of mitochondrial function and augmentation of glucose transport in vascular endothelial and smooth muscle cells.

The water-soluble and cell permeable nitroxide derivative 4-hydroxy tempol (TPL) has been shown to reduce or ameliorate oxidative stress-induced dysfunction and damage in vascular endothelial cells. We studied the effects of TPL on glucose transport and metabolism in bovine aortic endothelial (VEC) and smooth muscle cells (VSMC) under normal and high glucose conditions. Normally, these cells operate an autoregulatory protective mechanism that limits the rate of glucose transport under hyperglycemic conditions by decreasing the cell content of their typical glucose transporter GLUT-1 mRNA and protein as well as its plasma membrane abundance. TPL augmented the rate of glucose transport both under normo- and hyperglycemic conditions by increasing GLUT-1 mRNA and protein content and its plasma membrane abundance in both types of cells, leading to an increased flux of glucose into the cells. These effects were found related to ROS-generating and oxidant activities of TPL and to a decreased rate of mitochondrial ATP production under both normo- and hyperglycemic conditions. Since impaired mitochondrial functions, and in particular decreased rate of ATP production, augment the expression of GLUT-1 protein and glucose transport and metabolism, we suggest that the stimulatory effects of TPL in vascular cells results from its unfavorable interactions in the mitochondrion. It is therefore suggested that effects of TPL in cells of cardiovascular system be evaluated in parallel to its adverse effects on glucose and energy metabolism.