Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8-azido-AMP

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66394/1/j.1432-1033.1988.tb14407.x.pd

Mitochondrial Networking Protects β-Cells From Nutrient-Induced Apoptosis

OBJECTIVE: Previous studies have reported that β-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating β-cell mitochondrial morphology and function, remains unclear. In this article, we investigate β-cell mitochondrial fusion and fission in detail and report alterations in response to various combinations of nutrients. RESEARCH DESIGN AND METHODS: Using matrix-targeted photoactivatable green fluorescent protein, mitochondria were tagged and tracked in β-cells within intact islets, as isolated cells and as cell lines, revealing frequent fusion and fission events. Manipulations of key mitochondrial dynamics proteins OPA1, DRP1, and Fis1 were tested for their role in β-cell mitochondrial morphology. The combined effects of free fatty acid and glucose on β-cell survival, function, and mitochondrial morphology were explored with relation to alterations in fusion and fission capacity. RESULTS: β-Cell mitochondria are constantly involved in fusion and fission activity that underlies the overall morphology of the organelle. We find that networking activity among mitochondria is capable of distributing a localized green fluorescent protein signal throughout an isolated β-cell, a β-cell within an islet, and an INS1 cell. Under noxious conditions, we find that β-cell mitochondria become fragmented and lose their ability to undergo fusion. Interestingly, manipulations that shift the dynamic balance to favor fusion are able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis. CONCLUSIONS: These data suggest that alterations in mitochondrial fusion and fission play a critical role in nutrient-induced β-cell apoptosis and may be involved in the pathophysiology of type 2 diabetes.National Institutes of Health (R01HL071629-03, R01DK074778, 5T32DK007201

Tissue specific expression of human fatty acid oxidation enzyme genes in late pregnancy

Background: Abnormal fatty acid oxidation (FAO) is associated with maternal and fetal complications during pregnancy. The contribution of maternal and fetal tissues to FAO capacity during late pregnancy is important to understand the pathophysiology of pregnancy-associated complications. The aim of this study was to determine the expression levels of mitochondrial FAO enzymes in maternal and fetal tissues during late normal pregnancy. Methods: We have measured by Real-time PCR the levels of long- and medium -chain acyl-CoA dehydrogenase (LCHAD and MCAD), two acyl-CoA dehydrogenases that catalyze the initial step in the mitochondrial FAO spiral. Results: LCHAD and MCAD were expressed in maternal skeletal muscle, subcutaneous adipose tissue, placenta, and maternal and fetal blood cells. LCHAD gene expression was four- to 16-fold higher than MCAD gene expression in placenta, adipose tissue and skeletal muscle. In contrast, MCAD gene expression was ~5-fold higher in fetal blood than maternal blood (p = 0.02), whereas LCHAD gene expression was similar between fetal blood and maternal blood (p =0.91). Conclusions: LCHAD and MCAD are differentially expressed in maternal and fetal tissues during normal late pregnancy, which may represent a metabolic adaptation in response to physiological maternal dyslipidemia during late pregnancy.Consejeria de Salud, Junta de Andalucía Num Expte: 0269/05

Rh-POP Pincer Xantphos Complexes for C-S and C-H Activation. Implications for Carbothiolation Catalysis

The neutral Rh­(I)–Xantphos complex [Rh­(κ³-_P,O,P-Xantphos)­Cl]_<i>n</i>, <b>4</b>, and cationic Rh­(III) [Rh­(κ³-_P,O,P-Xantphos)­(H)₂]­[BAr^F₄], <b>2a</b>, and [Rh­(κ³-_P,O,P-Xantphos-3,5-C₆H₃(CF₃)₂)­(H)₂]­[BAr^F₄], <b>2b</b>, are described [Ar^F = 3,5-(CF₃)₂C₆H₃; Xantphos = 4,5-bis­(diphenylphosphino)-9,9-dimethylxanthene; Xantphos-3,5-C₆H₃(CF₃)₂ = 9,9-dimethylxanthene-4,5-bis­(bis­(3,5-bis­(trifluoromethyl)­phenyl)­phosphine]. A solid-state structure of <b>2b</b> isolated from C₆H₅Cl solution shows a κ¹-chlorobenzene adduct, [Rh­(κ³-_P,O,P-Xantphos-3,5-C₆H₃(CF₃)₂)­(H)₂(κ¹-ClC₆H₅)]­[BAr^F₄], <b>3</b>. Addition of H₂ to <b>4</b> affords, crystallographically characterized, [Rh­(κ³-_P,O,P-Xantphos)­(H)₂Cl], <b>5</b>. Addition of diphenyl acetylene to <b>2a</b> results in the formation of the C–H activated metallacyclopentadiene [Rh­(κ³-_P,O,P-Xantphos)­(ClCH₂Cl)­(σ,σ-(C₆H₄)­C­(H)CPh)]­[BAr^F₄], <b>7</b>, a rare example of a crystallographically characterized Rh–dichloromethane complex, alongside the Rh­(I) complex <i>mer</i>-[Rh­(κ³-_P,O,P-Xantphos)­(η²-PhCCPh)]­[BAr^F₄], <b>6</b>. Halide abstraction from [Rh­(κ³-_P,O,P-Xantphos)­Cl]_<i>n</i> in the presence of diphenylacetylene affords <b>6</b> as the only product, which in the solid state shows that the alkyne binds perpendicular to the κ³-POP Xantphos ligand plane. This complex acts as a latent source of the [Rh­(κ³-_P,O,P-Xantphos)]⁺ fragment and facilitates <i>ortho</i>-directed C–S activation in a number of 2-arylsulfides to give <i>mer</i>-[Rh­(κ³-_P,O,P-Xantphos)­(σ,κ¹-Ar)­(SMe)]­[BAr^F₄] (Ar = C₆H₄COMe, <b>8</b>; C₆H₄(CO)­OMe, <b>9</b>; C₆H₄NO₂, <b>10</b>; C₆H₄CNCH₂CH₂O, <b>11</b>; C₆H₄C₅H₄N, <b>12</b>). Similar C–S bond cleavage is observed with allyl sulfide, to give <i>fac</i>-[Rh­(κ³-_P,O,P-Xantphos)­(η³-C₃H₅)­(SPh)]­[BAr^F₄], <b>13</b>. These products of C–S activation have been crystallographically characterized. For <b>8</b> in situ monitoring of the reaction by NMR spectroscopy reveals the initial formation of <i>fac</i>-κ³-<b>8</b>, which then proceeds to isomerize to the <i>mer</i>-isomer. With the <i>para</i>-ketone aryl sulfide, 4-SMeC ₆H₄COMe, C–H activation <i>ortho</i> to the ketone occurs to give <i>mer</i>-[Rh­(κ³-_P,O,P-Xantphos)­(σ,κ¹-4-(COMe)­C₆H₃SMe)­(H)]­[BAr^F₄], <b>14</b>. The temporal evolution of carbothiolation catalysis using <i>mer</i>-κ³-<b>8</b>, and phenyl acetylene and 2-(methylthio)­acetophenone substrates shows initial fast catalysis and then a considerably slower evolution of the product. We suggest that the initially formed <i>fac</i>-isomer of the C–S activation product is considerably more active than the <i>mer</i>-isomer (i.e., <i>mer</i>-<b>8</b>), the latter of which is formed rapidly by isomerization, and this accounts for the observed difference in rates. A likely mechanism is proposed based upon these data

Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes

In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies

Per-arnt-sim (PAS) domain-containing protein kinase is downregulated in human islets in type 2 diabetes and regulates glucagon secretion.

AIMS/HYPOTHESIS: We assessed whether per-arnt-sim (PAS) domain-containing protein kinase (PASK) is involved in the regulation of glucagon secretion. METHODS: mRNA levels were measured in islets by quantitative PCR and in pancreatic beta cells obtained by laser capture microdissection. Glucose tolerance, plasma hormone levels and islet hormone secretion were analysed in C57BL/6 Pask homozygote knockout mice (Pask-/-) and control littermates. Alpha-TC1-9 cells, human islets or cultured E13.5 rat pancreatic epithelia were transduced with anti-Pask or control small interfering RNAs, or with adenoviruses encoding enhanced green fluorescent protein or PASK. RESULTS: PASK expression was significantly lower in islets from human type 2 diabetic than control participants. PASK mRNA was present in alpha and beta cells from mouse islets. In Pask-/- mice, fasted blood glucose and plasma glucagon levels were 25 ± 5% and 50 ± 8% (mean ± SE) higher, respectively, than in control mice. At inhibitory glucose concentrations (10 mmol/l), islets from Pask-/- mice secreted 2.04 ± 0.2-fold (p < 0.01) more glucagon and 2.63 ± 0.3-fold (p < 0.01) less insulin than wild-type islets. Glucose failed to inhibit glucagon secretion from PASK-depleted alpha-TC1-9 cells, whereas PASK overexpression inhibited glucagon secretion from these cells and human islets. Extracellular insulin (20 nmol/l) inhibited glucagon secretion from control and PASK-deficient alpha-TC1-9 cells. PASK-depleted alpha-TC1-9 cells and pancreatic embryonic explants displayed increased expression of the preproglucagon (Gcg) and AMP-activated protein kinase (AMPK)-alpha2 (Prkaa2) genes, implying a possible role for AMPK-alpha2 downstream of PASK in the control of glucagon gene expression and release. CONCLUSIONS/INTERPRETATION: PASK is involved in the regulation of glucagon secretion by glucose and may be a useful target for the treatment of type 2 diabetes

Combined Experimental and Computational Studies on the Nature of Aromatic C−H Activation by Octahedral Ruthenium(II) Complexes: Evidence for σ-Bond Metathesis from Hammett Studies

Octahedral ruthenium complexes of the type TpRu(L)(NCMe)R [Tp = hydridotris(pyrazolyl)borate; R = alkyl or aryl; L = CO or PMe3] have been shown previously to initiate the C-H activation of aromatic substrates. In order to probe the nature of the C-H activation step, reaction rates have been theoretically obtained for the conversion of TpRu(L)(η2-C, C-C6H5X)Me to TpRu(L)(P-C6H4X) and CH4 where X is varied among Br, Cl, CN, F, H, NH2, NO 2, and OMe. A linear Hammett correlation is calculated with a positive p value of 2.6 for L = CO and 3.2 for L = PMe3. Calculated kinetic data for the aromatic C-H activations indicate that an electrophilic aromatic substitution mechanism is unlikely. While experiments cannot fully replicate the entire range of calculated Hammett plots, reactivity trends are consistent with the calculations that suggest activation barriers to overall metal-mediated arene C-H bond cleavage are reduced by the presence of electron-withdrawing groups in the position para to the site of activation. Previous mechanistic studies, as well as the structure and imaginary vibrational modes of the present transition states, validate that the C-H activation for this family of TpRu complexes occurs through a σ-bond metathesis-type pathway. © 2007 American Chemical Society