Epoxygenase Cyp2c44 regulates hepatic lipid metabolism and insulin signaling by controlling FATP2 localization and activation of the DAG/PKCδ axis.

Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET) producing enzyme, promotes insulin sensitivity and Cyp2c44(-/-) mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SD) fed male Cyp2c44(-/-) mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared to wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol levels. Cyp2c44(-/-) mice had impaired glucose tolerance and increased hepatic plasma membraneassociated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SD and high fat diet fed (HFD) Cyp2c44(-/-) mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SD-fed Cyp2c44(-/-) mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44(-/-) hepatocytes. Finally, Cyp2c44(-/-) HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKDδ levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, diacylglycerol accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling.

A new ratiometric switch "two-way" detects hydrazine and hypochlorite via a "dye-release" mechanism with a PBMC bioimaging study.

A new ratiometric fluorescent probe (E)-2-(benzo[d]thiazol-2-yl)-3-(8-methoxyquinolin-2-yl)acrylonitrile (HQCN) was synthesised by the perfect blending of quinoline and a 2-benzothiazoleacetonitrile unit. In a mixed aqueous solution, HQCN reacts with hydrazine (N2H4) to give a new product 2-(hydrazonomethyl)-8-methoxyquinoline along with the liberation of the 2-benzothiazoleacetonitrile moiety. In contrast, the reaction of hypochlorite ions (OCl-) with the probe gives 8-methoxyquinoline-2-carbaldehyde. In both cases, the chemodosimetric approaches of hydrazine and hypochlorite selectively occur at the olefinic carbon but give two different products with two different outputs, as observed from the fluorescence study exhibiting signals at 455 nm and 500 nm for hydrazine and hypochlorite, respectively. A UV-vis spectroscopy study also depicts a distinct change in the spectrum of HQCN in the presence of hydrazine and hypochlorite. The hydrazinolysis of HQCN exhibits a prominent chromogenic as well as ratiometric fluorescence change with a 165 nm left-shift in the fluorescence spectrum. Similarly, the probe in hand (HQCN) can selectively detect hypochlorite in a ratiometric manner with a shift of 120 nm, as observed from the fluorescence emission spectra. HQCN can detect hydrazine and OCl- as low as 2.25 × 10-8 M and 3.46 × 10-8 M, respectively, as evaluated from the fluorescence experiments again. The excited state behaviour of the probe HQCN and the chemodosimetric products with hydrazine and hypochlorite are studied by the nanosecond time-resolved fluorescence technique. Computational studies (DFT and TDDFT) with the probe and the hydrazine and hypochlorite products were also performed. The observations made in the fluorescence imaging studies with human blood cells manifest that HQCN can be employed to monitor hydrazine and OCl- in human peripheral blood mononuclear cells (PBMCs). It is indeed a rare case that the single probe HQCN is found to be successfully able to detect hydrazine and hypochlorite in PBMCs, with two different outputs.

An excited state intramolecular proton transfer induced phosphate ion targeted ratiometric fluorescent switch to monitor phosphate ions in human peripheral blood mononuclear cells.

Detection of biological phosphate is very important for environmental and health care applications. In this study, a new ratiometric fluorescent probe (E)-N'-(3-(benzo[d]thiazol-2-yl)-2-hydroxybenzylidene) picolinohydrazide (BTP) is developed and exhibits a prominent excited-state intramolecular proton-transfer (ESIPT) mechanism. The probe BTP undergoes a unique phosphate induced hydrolytic reaction in mixed aqueous solution which produces a colorimetric change associated with a huge red-shift of ∼130 nm in the UV-visible absorption spectrum. Initially, BTP exhibits a strong fluorescence emission as the ESIPT process is 'on' and the tautomeric hydrogen remains flexible and is free to give two tautomeric forms. Eventually, after the addition of PO43-, the two tautomeric forms break and thereby shift the equilibrium towards the 'enol' form. The phosphate ion binds with BTP which is associated with a ratiometric change and accounts for an enhancement in the fluorescence intensity with a large blue shift and the limit of detection value of 8.33 × 10-8 M in a mixed aqueous medium. The binding constant (1.92 × 105 M-1) proportionally reflects the stability of the complexation between the binding sites of BTP with the guest PO43- anion. The probable mechanism is supported by the NMR spectroscopy studies. The sensing phenomenon is found to be reversible towards Zn2+ and thus the sensor beautifully mimics the INHIBIT logic gate. Observations have been made in fluorescence imaging studies with human peripheral blood mononuclear cells (PBMCs) which indicates that BTP can be employed to successfully monitor the phosphate ion in human PBMCs.

The soluble epoxide hydrolase inhibitor GSK2256294 decreases the proportion of adipose pro-inflammatory T cells.

Adipose tissue contains a complex immune environment and is a central contributor to heightened systemic inflammation in obese persons. Epoxyeicosatrienoic acids (EETs) are lipid signaling molecules that decrease inflammation in obese animals, but their effect on inflammation in humans is unknown. The enzyme soluble epoxide hydrolase (sEH) hydrolyzes EETs to less active diols, and we hypothesized that pharmacologic sEH inhibition would decrease adipose inflammation in obese individuals. We treated obese prediabetic adults with the sEH inhibitor GSK2256294 versus placebo in a crossover design, collected subcutaneous abdominal adipose tissue via lipoaspiration and characterized the tissue T cell profile. Treatment with GSK2256294 decreased the percentage of pro-inflammatory T cells producing interferon-gamma (IFNγ), but not interleukin (IL)-17A, and decreased the amount of secreted tumor necrosis factor-alpha (TNFα). Understanding the contribution of the EET/sEH pathway to inflammation in obesity could lead to new strategies to modulate adipose and systemic inflammation.

EET Analog Treatment Improves Insulin Signaling in a Genetic Mouse Model of Insulin Resistance.

We previously showed that global deletion of the cytochrome P450 epoxygenase Cyp2c44, a major epoxyeicosatrienoic acid (EET) producing enzyme in mice, leads to impaired hepatic insulin signaling resulting in insulin resistance. This finding led us to investigate whether administration of a water soluble EET analog restores insulin signaling in vivo in Cyp2c44(-/-) mice and investigated the underlying mechanisms by which this effect is exerted. Cyp2c44(-/-) mice treated with the analog EET-A for 4 weeks improved fasting glucose and glucose tolerance compared to Cyp2c44(-/-) mice treated with vehicle alone. This beneficial effect was accompanied by enhanced hepatic insulin signaling, decreased expression of gluconeogenic genes and increased expression of glycogenic genes. Mechanistically, we show that insulin-stimulated phosphorylation of insulin receptor ß (IRß) is impaired in primary Cyp2c44(-/-) hepatocytes and this can be restored by cotreatment with EET-A and insulin. Plasma membrane fractionations of livers indicated that EET-A enhances the retention of IRß in membrane rich fractions, thus potentiating its activation. Altogether, EET analogs ameliorate insulin signaling in a genetic model of hepatic insulin resistance by stabilizing membrane-associated IRß and potentiating insulin signaling.

EET Analog Treatment Improves Insulin Signaling in a Genetic Mouse Model of Insulin Resistance.

We previously showed that global deletion of the cytochrome P450 epoxygenase Cyp2c44, a major epoxyeicosatrienoic acid (EET) producing enzyme in mice, leads to impaired hepatic insulin signaling resulting in insulin resistance. This finding led us to investigate whether administration of a water soluble EET analog restores insulin signaling in vivo in Cyp2c44(-/-) mice and investigated the underlying mechanisms by which this effect is exerted. Cyp2c44(-/-) mice treated with the analog EET-A for 4 weeks improved fasting glucose and glucose tolerance compared to Cyp2c44(-/-) mice treated with vehicle alone. This beneficial effect was accompanied by enhanced hepatic insulin signaling, decreased expression of gluconeogenic genes and increased expression of glycogenic genes. Mechanistically, we show that insulin-stimulated phosphorylation of insulin receptor ß (IRß) is impaired in primary Cyp2c44(-/-) hepatocytes and this can be restored by cotreatment with EET-A and insulin. Plasma membrane fractionations of livers indicated that EET-A enhances the retention of IRß in membrane rich fractions, thus potentiating its activation. Altogether, EET analogs ameliorate insulin signaling in a genetic model of hepatic insulin resistance by stabilizing membrane-associated IRß and potentiating insulin signaling.

Adiponectin Genetic Variant and Expression Coupled with Lipid Peroxidation Reveal New Signatures in Diabetic Dyslipidemia.

Increasing burden of non-communicable diseases like diabetes and cardiovascular disorders has made the global health scenario more challenging. Dyslipidemia in diabetes is a compounding risk factor for cardiovascular diseases, but there is dearth of identifying appropriate signatures to address this issue. The protein, adiponectin, is actively involved in regulating glucose levels as well as fatty acid breakdown playing crucial role in dyslipidemia and vascular complications. To identify the underlying genetic and molecular profile of adiponectin metabolic pathway in diabetic dyslipidemia and to correlate it with known biochemical and oxidative stress parameters of T2DM, we performed a case-control study in a total 264 individuals belonging to three categories such as diabetes with dyslipidemia (n = 88), diabetes without dyslipidemia (n = 86) and normal healthy controls (n = 90). Expression of adiponectin (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) were measured in visceral and subcutaneous adipose tissues. A significant downregulated expression of ADIPOQ and its receptors in adipose tissues and PBMCs were linked with diabetic dyslipidemic condition. A multiple linear regression followed by MDR analysis implicated the elevated plasma malondialdehyde and decreased adiponectin level to be correlated with diabetic dyslipidemia. More interestingly, two single nucleotide polymorphisms of ADIPOQ (rs2241766 and rs1501299) were genetically associated with the risk of developing dyslipidemia. Other important biochemical factors found to be increased in diabetic dyslipidemic conditions included plasma C-reactive protein and 4-hydroxynonenal adducts. Our results explore, a complex interplay of genetic and biochemical parameters in diabetic dyslipidemia which is significant from the perspective of risk stratification and novel therapeutic strategy development.

Treatment of Primary Aldosteronism Increases Plasma Epoxyeicosatrienoic Acids.

[Figure: see text].

Dielectric properties of plasma membrane: A signature for dyslipidemia in diabetes mellitus.

Dielectric properties of a living biological membrane play crucial role indicating the status of the cell in pathogenic or healthy condition. A distinct variation in membrane capacitance and impedance was observed for peripheral blood mononuclear cell (PBMC) suspensions for diabetic and diabetic-dyslipidemic subjects compared to healthy control. Low frequency region were explicitly considered in electrical analysis to address complex membrane dielectric factors that alter the system capacitance of a PBMC suspension. Such variation was marked in size, morphology and membrane function of PBMCs for control and diseased cases. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies reveal significant alteration in surface morphology of PBMCs in diseased condition. Side scatter of flow cytometry reveals complexity of PBMCs in diseased condition. Changes in size between groups were not found by SEM and forward scatter. Functional alteration in PBMCs was manifested by significant changes in cell membrane properties like Na+, K+ ATPase and Ca2+, Mg2+ ATPase activity, reduced plasma membrane fluidity and changes in intracellular Ca2+ content, which bear significant correlation in diabetic and diabetic dyslipidemic subjects. Therefore, dielectric parameters of PBMCs in diabetic-dyslipidemic challenges may led to interesting correlation opening the possibility of identifying crucial signature biomarkers.

Detection of HSO3-: A Rapid Colorimetric and Fluorimetric Selective Sensor for Detecting Biological SO2 in Food and Living Cells.

A rapid fluorescent probe based on the conjugate of chromone and benzothiazole moiety was presented, which could selectively respond to HSO3- over other common anions and thiols. The function of the probe relies on nucleophilic addition to break down the π-conjugation. The probe can be used as a signal tool to determine HSO3- levels in sugar-based food and living cells.

A novel sensor to estimate the prevalence of hypochlorous (HOCl) toxicity in individuals with type 2 diabetes and dyslipidemia.

BACKGROUND: Oxidative stress is common in type 2 diabetes. It is characterised by increased levels of reactive oxygen species (ROS), of which hypochlorous acid (HOCl) is an important component. Type 2 diabetes is characterised not only by hyperglycemia, but also by dyslipidemia. It probably underlies both the development of diabetes and also resulting complications, like cardiovascular disease. We applied a novel fluoroprobe RHQ to estimate endogenous HOCl and myeloperoxidase (MPO) activity in diabetes and dyslipidemia. METHODS: Our newly designed probe, RHQ (rhodamine-quinoline based chemodosimeter) is capable of estimating endogenous HOCl selectively out of the ROS components. Isolated leukocytes from study subjects were treated with DCFDA and monocytes and neutrophils with RHQ for estimating endogenous ROS and HOCl respectively. Plasma AOPP, an indicator of HOCl was also measured. We attempted to find out the key reasons of higher HOCl content in diabetic dyslipidemic subjects by quantitating endogenous hydrogen peroxide (H2O2) and myeloperoxidase (MPO) activity. RESULTS: Isolated PBMCs from diabetic dyslipidemic subjects indicated enhanced ROS and HOCl generation followed by diabetic subjects without dyslipidemia and healthy controls. We explored increased production of H2O2 and enhanced enzymatic activity of myeloperoxidase (MPO) among diabetic dyslipidemic subjects (p<0.0001) resulting in higher HOCl content. CONCLUSION: The hyperglycemic and hyperlipidemic challenges together enhance the production of HOCl and the fluoroprobe RHQ may be used as a novel diagnostic marker to evaluate the extent of this toxicity.

Adiponectin: Probe of the molecular paradigm associating diabetes and obesity.

Type 2 diabetes is an emerging health challenge all over the world as a result of urbanization, high prevalence of obesity, sedentary lifestyle and other stress related factors compounded with the genetic prevalence. The health consequences and economic burden of the obesity and related diabetes mellitus epidemic are enormous. Different signaling molecules secreted by adipocytes have been implicated in the development of obesity and associated insulin resistance in type 2 diabetes. Human adiponectin, a 244-amino acid collagen-like protein is solely secreted by adipocytes and acts as a hormone with anti-inflammatory and insulin-sensitizing properties. Adiponectin secretion, in contrast to secretion of other adipokines, is paradoxically decreased in obesity which may be attributable to inhibition of adiponectin gene transcription. There are several mechanisms through which adiponectin may decrease the risk of type 2 diabetes, including suppression of hepatic gluconeogenesis, stimulation of fatty acid oxidation in the liver, stimulation of fatty acid oxidation and glucose uptake in skeletal muscle, and stimulation of insulin secretion. To date, no systematic review has been conducted that evaluate the potential importance of adiponectin metabolism in insulin resistance. In this review attempt has been made to explore the relevance of adiponectin metabolism for the development of diabetes mellitus. This article also identifies this novel target for prospective therapeutic research aiming successful management of diabetes mellitus.

A triphenyl amine-based solvatofluorochromic dye for the selective and ratiometric sensing of OCl- in human blood cells.

A new visible-light-excitable fluorescence ratiometric probe for OCl(-) has been developed based on a triphenylamine-diamiomaleonitrile (TAM) moiety. The structure of the dye was confirmed by single-crystal X-ray analysis. It behaves as a highly selective and sensitive probe for OCl(-) over other analytes with a fast response time (∼100 s). OCl(-) reacts with the probe leading to the formation of the corresponding aldehyde in a mixed-aqueous system. The detection limit of the probe is in the 10(-8) M range. The probe (TAM) also exhibits solvatofluorochromism. Changing the solvent from non-polar to polar, the emission band of TAM largely red-shifted. Moreover, the probe shows an excellent performance in real-life application in detecting OCl(-) in human blood cells. The experimentally observed changes in the structure and electronic properties of the probe after reaction with OCl(-) were studied by DFT and TDDFT computational calculations.

Overview of platelet physiology: its hemostatic and nonhemostatic role in disease pathogenesis.

Platelets are small anucleate cell fragments that circulate in blood playing crucial role in managing vascular integrity and regulating hemostasis. Platelets are also involved in the fundamental biological process of chronic inflammation associated with disease pathology. Platelet indices like mean platelets volume (MPV), platelets distributed width (PDW), and platelet crit (PCT) are useful as cheap noninvasive biomarkers for assessing the diseased states. Dynamic platelets bear distinct morphology, where α and dense granule are actively involved in secretion of molecules like GPIIb , IIIa, fibrinogen, vWf, catecholamines, serotonin, calcium, ATP, ADP, and so forth, which are involved in aggregation. Differential expressions of surface receptors like CD36, CD41, CD61 and so forth have also been quantitated in several diseases. Platelet clinical research faces challenges due to the vulnerable nature of platelet structure functions and lack of accurate assay techniques. But recent advancement in flow cytometry inputs huge progress in the field of platelets study. Platelets activation and dysfunction have been implicated in diabetes, renal diseases, tumorigenesis, Alzheimer's, and CVD. In conclusion, this paper elucidates that platelets are not that innocent as they keep showing and thus numerous novel platelet biomarkers are upcoming very soon in the field of clinical research which can be important for predicting and diagnosing disease state.