Direct analysis of PI(3,4,5)P3 using liquid chromatography electrospray ionization tandem mass spectrometry.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a biologically active membrane phospholipid that is essential for the growth and survival of all eukaryotic cells. We describe a new method that directly measures PIP3 and describe the HPLC separation and measurement of the positional isomers of phosphatidylinositol bisphosphate, PI(3,5)P2, PI(3,4)P2 and PI(4,5)P2. Mass spectrometric analyses were performed online using ultra-high performance liquid chromatography (UHPLC)-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the negative multiple-reaction monitoring (MRM) modes. Rapid separation of PIP3 from PI, phosphatidylinositol phosphate (PIP) and PIP2 was accomplished by C18 reverse phase chromatography with the addition of the ion pairing reagents diisopropylethanolamine (DiiPEA) and ethylenediamine tetraacetic acid tetrasodium salt dihydrate (EDTA) to the samples and mobile phase with a total run time, including equilibration, of 12 min. Importantly, these chromatography conditions result in no carryover of PIP, PIP2, and PIP3 between samples. To validate the new method, U87MG cancer cells were serum starved and treated with PDGF to stimulate PIP3 biosynthesis in the presence or absence of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Results generated with the LC/MS method were in excellent agreement with results generated using [33P] phosphate radiolabeled U87MG cells and anion exchange chromatography analysis, a well validated method for measuring PIP3. To demonstrate the usefulness of the new method, we generated reproducible IC50 data for several well-characterized PI3K small molecule inhibitors using a U87MG cell-based assay as well as showing PIP3 can be measured from additional cancer cell lines. Together, our results demonstrate this novel method is sensitive, reproducible and can be used to directly measure PIP3 without radiolabeling or complex lipid derivatization.

Synthesis and Characterization of Urofuranoic Acids: In Vivo Metabolism of 2-(2-Carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic Acid (CMPF) and Effects on in Vitro Insulin Secretion.

CMPF (2-(2-carboxyethyl)-4-methyl-5-propylfuran-3-carboxylic acid) is a metabolite that circulates at high concentrations in type 2 and gestational diabetes patients. Further, human clinical studies suggest it might have a causal role in these diseases. CMPF inhibits insulin secretion in mouse and human islets in vitro and in vivo in rodents. However, the metabolic fate of CMPF and the relationship of structure to effects on insulin secretion have not been significantly studied. The syntheses of CMPF and analogues are described. These include isotopically labeled molecules. Study of these materials in vivo has led to the first observation of a metabolite of CMPF. In addition, a wide range of CMPF analogues have been prepared and characterized in insulin secretion assays using both mouse and human islets. Several molecules that influence insulin secretion in vitro were identified. The molecules described should serve as interesting probes to further study the biology of CMPF.

MitoNEET-Parkin Effects in Pancreatic α- and ß-Cells, Cellular Survival, and Intrainsular Cross Talk.

Mitochondrial metabolism plays an integral role in glucose-stimulated insulin secretion (GSIS) in ß-cells. In addition, the diabetogenic role of glucagon released from α-cells plays a major role in the etiology of both type 1 and type 2 diabetes because unopposed hyperglucagonemia is a pertinent contributor to diabetic hyperglycemia. Titrating expression levels of the mitochondrial protein mitoNEET is a powerful approach to fine-tune mitochondrial capacity of cells. Mechanistically, ß-cell-specific mitoNEET induction causes hyperglycemia and glucose intolerance due to activation of a Parkin-dependent mitophagic pathway, leading to the formation of vacuoles and uniquely structured mitophagosomes. Induction of mitoNEET in α-cells leads to fasting-induced hypoglycemia and hypersecretion of insulin during GSIS. MitoNEET-challenged α-cells exert potent antiapoptotic effects on ß-cells and prevent cellular dysfunction associated with mitoNEET overexpression in ß-cells. These observations identify that reduced mitochondrial function in α-cells exerts potently protective effects on ß-cells, preserving ß-cell viability and mass.

Reducing plasma membrane sphingomyelin increases insulin sensitivity.

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.

Studies on the chemical reactivity of diclofenac acyl glucuronide with glutathione: identification of diclofenac-S-acyl-glutathione in rat bile.

Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to a reactive acyl glucuronide that has been proposed to mediate toxic adverse drug reactions associated with its use. In the present study, we examined the ability of diclofenac acyl glucuronide (D-1-O-G) to transacylate glutathione (GSH) in vitro in buffer and in vivo in rats. Thus, in vitro reactions of D-1-O-G (100 microM) with GSH (10 mM) at pH 7.4 and 37 degrees C showed a linear time-dependent formation of diclofenac-S-acyl-glutathione (D-SG, 3 microM/h) through 60 min of incubation, reaching a maximum of 3.7 microM after 2 h of incubation. The major reaction that occurred was acyl migration of D-1-O-G (t1/2, 54 min) to less reactive isomers. The D-SG thioester product was shown to be unstable by degrading primarily to 1-(2,6-dichlorophenyl)indolin-2-one and by hydrolysis to diclofenac. After administration of diclofenac to rats (200 mg/kg), bile was collected and analyzed for D-SG by liquid chromatography-tandem mass spectrometry. Results indicated the presence of D-SG, which was confirmed by coelution with synthetic standard and by its tandem mass spectrum. When the reactivity of D-SG (100 microM) was compared with D-1-O-G (100 microM) in vitro in reactions with N-acetylcysteine (NAC, 10 mM), results showed the quantitative reaction of D-SG with NAC after 30 min of incubation, whereas only approximately 1% of D-1-O-G reacted to form diclofenac-S-acyl-NAC at the same time point. Results from these studies indicate that GSH reacts with D-1-O-G in vitro, and presumably in vivo, to form D-SG, and that the product D-SG thioester is chemically more reactive in transacylation-type reactions than the D-1-O-G metabolite.