Ileal interposition (IT) surgery delays the onset of diabetes in a rat model of type-2 diabetes (UCD-T2DM). Here, to gain a deeper understanding of the molecular events underlying the effects of IT surgery, we examined the changes in the proteome of four white adipose depots (retroperitoneal, mesenteric, inguinal, and epididymal) and plasma-free fatty acid profile in pre-diabetic rats 1.5 months following IT or sham surgery. The IT-mediated changes were exerted mainly in mesenteric fat and spanned from delayed adipocyte maturation to a neuroendocrine remodeling. Conversely, inguinal, retroperitoneal, and epididymal depots showed opposite trends consistent with increased adipocyte maturation and adipogenesis development prior to overt signs of diabetes, probably orchestrated by peroxisome proliferator-activated receptor gamma signaling and higher plasma n -6/ n -3 free fatty acid ratios. The resulting scenario suggests a targeted use of surgical strategies that seek to delay or improve diabetes in order to manipulate adipose depot-specific responses to maximize the duration and beneficial effects of the surgery.

A multitude of natural and artificial compounds have been recognized to modulate autophagy, providing direct or, through associated pathways, indirect entry points to activation and inhibition. While these pharmacological tools are extremely useful in the study of autophagy, their abundance also suggests the potential presence of unidentified autophagic modulators that may interfere with experimental designs if applied unknowingly. Here, we report unanticipated effects on autophagy and bioenergetics in neuronal progenitor cells (NPCs) incubated with the widely used lipid-based transfection reagent lipofectamine (LF), which induced mitochondria depolarization followed by disruption of electron transport. When NPCs were exposed to LF for 5 h followed by 24, 48, and 72 h in LF-free media, an immediate increase in mitochondrial ROS production and nitrotyrosine formation was observed. These events were accompanied by disrupted mitophagy (accumulation of dysfunctional and damaged mitochondria, and of LC3II and p62), in an mTOR- and AMPK-independent manner, and despite the increased mitochondrial PINK1 (PTEN-inducible kinase 1) localization. Evidence supported a role for a p53-mediated abrogation of parkin translocation and/or abrogation of membrane fusion between autophagosome and lysosomes. While most of the outcomes were LF-specific, only two were shared by OptiMEM exposure (with no serum and reduced glucose levels) albeit at lower extents. Taken together, our findings show that the use of transfection reagents requires critical evaluation with respect to consequences for overall cellular health, particularly in experiments designed to address autophagy-inducing effects and/or energy stress.

Carriers of premutation CGG expansions in the fragile X mental retardation 1 ( FMR1 ) gene are at higher risk of developing a late-onset neurodegenerative disorder named Fragile X-associated tremor ataxia syndrome (FXTAS). Given that mitochondrial dysfunction has been identified in fibroblasts, PBMC and brain samples from carriers as well as in animal models of the premutation and that mitochondria are at the center of intermediary metabolism, the aim of the present study was to provide a complete view of the metabolic pattern by uncovering plasma metabolic perturbations in premutation carriers. To this end, metabolic profiles were evaluated in plasma from 23 premutation individuals and 16 age- and sex-matched controls. Among the affected pathways, mitochondrial dysfunction was associated with a Warburg-like shift with increases in lactate levels and altered Krebs' intermediates, neurotransmitters, markers of neurodegeneration and increases in oxidative stress-mediated damage to biomolecules. The number of CGG repeats correlated with a subset of plasma metabolites, which are implicated not only in mitochondrial disorders but also in other neurological diseases, such as Parkinson's, Alzheimer's and Huntington's diseases. For the first time, the identified pathways shed light on disease mechanisms contributing to morbidity of the premutation, with the potential of assessing metabolites in longitudinal studies as indicators of morbidity or disease progression, especially at the early preclinical stages.

Insulin-like peptides (ILPs) play important roles in growth and metabolic homeostasis, but have also emerged as key regulators of stress responses and immunity in a variety of vertebrates and invertebrates. Furthermore, a growing literature suggests that insulin signaling-dependent metabolic provisioning can influence host responses to infection and affect infection outcomes. In line with these studies, we previously showed that knockdown of either of two closely related, infection-induced ILPs, ILP3 and ILP4, in the mosquito Anopheles stephensi decreased infection with the human malaria parasite Plasmodium falciparum through kinetically distinct effects on parasite death. However, the precise mechanisms by which ILP3 and ILP4 control the response to infection remained unknown. To address this knowledge gap, we used a complementary approach of direct ILP supplementation into the blood meal to further define ILP-specific effects on mosquito biology and parasite infection. Notably, we observed that feeding resulted in differential effects of ILP3 and ILP4 on blood-feeding behavior and P. falciparum development. These effects depended on ILP-specific regulation of intermediary metabolism in the mosquito midgut, suggesting a major contribution of ILP-dependent metabolic shifts to the regulation of infection resistance and parasite transmission. Accordingly, our data implicate endogenous ILP signaling in balancing intermediary metabolism for the host response to infection, affirming this emerging tenet in host–pathogen interactions with novel insights from a system of significant public health importance.

FXTAS (fragile X-associated tremor/ataxia syndrome) is a late-onset neurodegenerative disorder that affects individuals who are carriers of premutation expansions (55–200 CGG repeats) in the 5′ untranslated region of the FMR1 (fragile X mental retardation 1) gene. The role of MD (mitochondrial dysfunction) in FXTAS was evaluated in fibroblasts and brain samples from premutation carriers with and without FXTAS symptoms, with a range of CGG repeats. This study resulted in several important conclusions: (i) decreased NAD- and FAD-linked oxygen uptake rates and uncoupling between electron transport and synthesis of ATP were observed in fibroblasts from premutation carriers; (ii) a lower expression of mitochondrial proteins preceded both in age and in CGG repeats the appearance of overt clinical involvement; (iii) the CGG repeat size required for altered mitochondrial protein expression was also smaller than that required to produce brain intranuclear inclusions from individuals with the premutation who died, suggesting that MD is an incipient pathological process occurring in individuals who do not display overt features of FXTAS; and (iv) on the basis of the CGG repeats, MD preceded the increase in oxidative/nitrative stress damage, indicating that the latter is a late event. MD in carriers of small CGG repeats, even when the allele size is not sufficient to produce FXTAS, may predispose them to other disorders (e.g. Parkinson's disease) that are likely to involve MD, and to environmental stressors, which may trigger the development of FXTAS symptoms. Detection of MD is of critical importance to the management of FXTAS, since it opens up additional treatment options for this disorder.

Tyrosine nitration is a covalent post-translational protein modification associated with various diseases related to oxidative/nitrative stress. A role for nitration of tyrosine in protein inactivation has been proposed; however, few studies have established a direct link between this modification and loss of protein function. In the present study, we determined the effect of nitration of Tyr 345 and Tyr 368 in the β-subunit of the F 1 -ATPase using site-directed mutagenesis. Nitration of the β-subunit, achieved by using TNM (tetranitromethane), resulted in 66% ATPase activity loss. This treatment resulted in the modification of several asparagine, methionine and tyrosine residues. However, nitrated tyrosine and ATPase inactivation were decreased in reconstituted F 1 with Y368F (54%), Y345F (28%) and Y345,368F (1%) β-subunits, indicating a clear link between nitration at these positions and activity loss, regardless of the presence of other modifications. Kinetic studies indicated that an F 1 with one nitrated tyrosine residue (Tyr 345 or Tyr 368 ) or two Tyr 368 residues was sufficient to grant inactivation. Tyr 368 was four times more reactive to nitration due to its lower p K a . Inactivation was attributed mainly to steric hindrance caused by adding a bulky residue more than the presence of a charged group or change in the phenolic p K a due to the introduction of a nitro group. Nitration at this residue would be more relevant under conditions of low nitrative stress. Conversely, at high nitrative stress conditions, both tyrosine residues would contribute equally to ATPase inactivation.

No studies have been performed on the mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that have a significant impact on malaria parasite transmission in endemic regions. In the present study, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells [ASE ( Anopheles stephensi Mos. 43) cell line] from A. stephensi , a major vector of malaria in India, South-East Asia and parts of the Middle East. ASE cell mitochondria share many features in common with mammalian muscle mitochondria, despite the fact that these cells are of larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol–phosphate shuttle plays as major a role in NADH oxidation in ASE cell mitochondria as it does in insect muscle mitochondria. In contrast, mammalian white muscle mitochondria depend primarily on lactate dehydrogenase, whereas red muscle mitochondria depend on the malate–oxaloacetate shuttle. Two, ASE mitochondria were able to oxidize proline at a rate comparable with that of α-glycerophosphate. However, the proline pathway appeared to differ from the currently accepted pathway, in that oxoglutarate could be catabolized completely by the tricarboxylic acid cycle or via transamination, depending on the ATP need.

S-nitrosation of protein thiol groups by nitric oxide (NO • ) is a widely recognized protein modification. Only few intracellular S-nitrosated proteins have been identified and it has been reported that S-nitrosation/denitrosation can serve as a regulatory process in signal-transduction pathways. Given the potential physiological importance of S -nitrosothiols, and considering that mitochondria are endowed with high levels of thiols and the biochemical requisites for synthesizing NO • , we examined the occurrence of S -nitrosoglutathione (GSNO) in intact, coupled rat liver mitochondria. These organelles contained 0.34nmol of GSNO/mg of protein, detected by HPLC with UV–visible and electrochemical detections. This concentration was dynamically modulated by the availability of NO • ; its decay was affected mainly by GSH and superoxide dismutase in a reaction that entailed the generation of GSSG. On the basis of the relatively long half-life of GSNO and the negligible recovery of NO • during its decay, roles for GSNO as a storage and transport molecule for NO • are discussed. Moreover, the formation of GSNO and its reaction with GSH can be considered to be partly responsible for the catabolism of NO • via a complex mechanism that might result in the formation of hydroxylamine, nitrite or nitrous oxide depending upon the availability of oxygen, superoxide dismutase and glutathione. Finally, the high concentrations of GSH in the cytosol and mitochondria might favour the formation of GSNO by reacting with NO • ‘in excess’, thereby avoiding damaging side reactions (such as peroxynitrite formation), and facilitate the inactivation of NO • by generating other nitrogen-related species without the chemical properties characteristic of NO • .

The effects of endogenous production of NO • , catalysed by the mitochondrial nitric oxide synthase (NOS), on mitochondrial metabolism were studied. The respiratory rates of intact mitochondria in State 4 were decreased by 40% and 28% with succinate and malate–glutamate, respectively, in the presence of l -arginine ( l -Arg); conversely, the O 2 uptake with N G - methyl- l -arginine (NMMA), a competitive inhibitor of NOS, was increased. The production of NO • and the inhibition of the respiratory rates were dependent on the metabolic state in which mitochondria were maintained: NO • production was probably supported by mitochondrial NADPH, the latter maintained by the energy-dependent transhydrogenase. In addition to the decline in the respiratory rate, an inhibition of ATP synthesis was also observed (40–50%) following supplementation with l -Arg. The dependence of the respiratory rates of mitochondria in State 3 and cytochrome oxidase activities on O 2 concentrations with either l -Arg or NMMA indicated that both processes were competitively inhibited by NO • at the cytochrome oxidase level. This inhibition can be explained by the interaction of NO • with cytochrome oxidase at the binuclear centre. The role of NO • as a physiological modulator of cytochrome oxidase is discussed in terms of cellular metabolism.