Cellular localization and associations of the major lipolytic proteins in human skeletal muscle at rest and during exercise

Lipolysis involves the sequential breakdown of fatty acids from triacylglycerol and is increased during energy stress such as exercise. Adipose triglyceride lipase (ATGL) is a key regulator of skeletal muscle lipolysis and perilipin (PLIN) 5 is postulated to be an important regulator of ATGL action of muscle lipolysis. Hence, we hypothesized that non-genomic regulation such as cellular localization and the interaction of these key proteins modulate muscle lipolysis during exercise. PLIN5, ATGL and CGI-58 were highly (>60%) colocated with Oil Red O (ORO) stained lipid droplets. PLIN5 was significantly colocated with ATGL, mitochondria and CGI-58, indicating a close association between the key lipolytic effectors in resting skeletal muscle. The colocation of the lipolytic proteins, their independent association with ORO and the PLIN5/ORO colocation were not altered after 60 min of moderate intensity exercise. Further experiments in cultured human myocytes showed that PLIN5 colocation with ORO or mitochondria is unaffected by pharmacological activation of lipolytic pathways. Together, these data suggest that the major lipolytic proteins are highly expressed at the lipid droplet and colocate in resting skeletal muscle, that their localization and interactions appear to remain unchanged during prolonged exercise, and, accordingly, that other post-translational mechanisms are likely regulators of skeletal muscle lipolysis

BAFF and MyD88 signals promote a lupuslike disease independent of T cells

Systemic lupus erythernatosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. However, the underlying cause of disease appears to relate to defects in T cell tolerance or T cell help to 13 cells. Transgenic (Tg) mice over-expressing the cytokine 13 cell-activating factor of the tumor necrosis factor family (BAFF) develop an autoimmune disorder similar to SLE and show impaired B cell tolerance and altered T cell differentiation. We generated BAFF Tg mice that were completely deficient in T cells, and, surprisingly, these mice developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did, however, require 13 cell-intrinsic signals through the Toll-like receptor (TLR)-associated signaling adaptor MyD88, which controlled the production of proinflammatory autoantibody isotypes. TLR7/9 activation strongly up-regulated expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which is a receptor for BAFF involved in 13 cell responses to T cell-independent antigens. Moreover, BAFF enhanced TLR7/9 expression on 13 cells and TLR-mediated production of autoantibodies. Therefore, autoirnmunity in BAFF Tg mice results from altered 13 cell tolerance, but requires TLR signaling and is independent of T cell help. It is possible that SLE patients with elevated levels of BAFF show a similar basis for disease

PLIN5 deletion remodels intracellular lipid composition and causes insulin resistance in muscle

Defective control of lipid metabolism leading to lipotoxicity causes insulin resistance in skeletal muscle, a major factor leading to diabetes. Here, we demonstrate that perilipin (PLIN) 5 is required to couple intramyocellular triacylglycerol lipolysis with the metabolic demand for fatty acids. PLIN5 ablation depleted triacylglycerol stores but increased sphingolipids including ceramide, hydroxylceramides and sphingomyelin. We generated perilipin 5 (Plin5)-/- mice to determine the functional significance of PLIN5 in metabolic control and insulin action. Loss of PLIN5 had no effect on body weight, feeding or adiposity but increased whole-body carbohydrate oxidation. Plin5-/- mice developed skeletal muscle insulin resistance, which was associated with ceramide accumulation. Liver insulin sensitivity was improved in Plin5-/- mice, indicating tissue-specific effects of PLIN5 on insulin action. We conclude that PLIN5 plays a critical role in coordinating skeletal muscle triacylglycerol metabolism, which impacts sphingolipid metabolism, and is requisite for the maintenance of skeletal muscle insulin action. © 2014 The Authors

Pigmentsynthese bei Exophiala dermatitidis : Einfluss auf Phagozytose und Oxidative Burst

Indolderivate wurden von der Arbeitsgruppe um Prof. Mayser 1998 erstmals bei der Hefe Malassezia furfur beschrieben, bei der sie vermutlich in einem klinischen Zusammenhang mit der Hautkrankheit Pityriasis versicolor stehen. Im Jahr 2006 konnten Nies et al. solche Indolderivate erstmals bei Exophiala dermatitidis nachweisen. Ziel der vorliegenden Arbeit war die vergleichende Untersuchung der Pigmentspektren verschiedener Stämme von Exophiala dermatitidis und ihre Überprüfung auf eine mögliche Rolle in der Pathogenese von Exophiala-dermatitidis-Infektionen. Hierfür wurden der Wildtyp und seine Mutanten Mel-1, Mel-3 und Mel-4 zur Pigmentsynthese auf 1b-Minimalagar angezüchtet. Nach vierwöchiger Wachstumsphase wurde das dabei entstandene Pigment als Rohextrakt gewonnen, durch chromatographische Methoden vorgereinigt, sowie einzelne farbige Bestandteile weiter aufgereinigt. Die Stukturaufklärung isolierter Reinsubstanzen erfolgte an der TU München. Grundlage zur Untersuchung des Oxidative Burst stellte der von Schnitzler et al. (1999) etablierte Vollblutansatz mit Dihydrorhodamine 123 als Indikator für die Sauerstoffradikalsynthese von neutrophilen Granulozyten dar. Die anschließende Messung erfolgte am Durchflußzytometer. Im Rahmen der Untersuchung der Pigmentspektren konnte beim Wildtyp von Exophiala dermatitidis erstmals die Synthese von Pityriacitrin, Pityriarubin C, Exophialin und Desoxyexophialin nachgewiesen werden. Für die Mutante Mel-1 konnte erstmals das bislang nur durch vergleichende DC nachgewiesene Pityriacitrin in der HPLC, sowie durch Analyse der TU München bestätigt werden. Desweiteren konnte Exophialin aus zwei violetten Banden mit einem Rf-Wert von 0,38 und 0,56 isoliert werden. Unter den Syntheseprodukten der Mutante Mel-3 wurde erstmals Exophialin, Pityriarubin C und Pityriacitrin nachgewiesen. Beim Vergleich des Wildtyps von Exophiala dermatitidis mit seiner Melaninmangel-Mutante Mel-3 hinsichtlich ihrer Auswirkung auf den Oxidative Burst in Vollblutproben konnte eine deutliche Hemmung des Oxidative Burst durch den Wildtyp gegenüber Mel-3 aufgezeigt werden. Dies läßt den Rückschluß auf eine deutliche Schutzwirkung des vom Wildtyp synthetisierten Melanins vor der ROS-Synthese zu. Auch die untersuchten Pigment-Rohextrakte von Wildtyp, Mel-1, Mel-3 und Mel-4 hatten alle eine hemmende Wirkung auf die ROS-Synthese, wobei der Rohextrakt von Mel-3 den stärksten Einfluß hatte. Bei den Sephadex-Fraktionen der Mutante Mel-3 ergaben unterschiedliche Einflüsse auf die ROS-Synthese. Die Fraktionen zwei und sechs zeigten in allen Zeitstufen zwischen 0 und 60 Minuten die stärkste Unterdrückung, wohingegen bei den Fraktionen drei und acht zu keinem Zeitpunkt Auswirkungen auf die ROS-Synthese beobachtet wurden. Nach weiterer Unterteilung der hemmenden Fraktion sechs der Mutante Mel-3 mittels präparativer Dünnschichtchromatographie in acht Banden konnte eine deutliche Hemmung des Oxidative Burst durch die Banden 5-8 verzeichnet werden. In diesem Bereich kam neben zahlreichen Fluorochromen und Pityriarubin C eine prominente Bande von grauer Farbe vor. Diese Substanz hemmte die ROS-Synthese deutlich, wohingegen Pityriarubin C keinen Einfluß darauf hatte. Auch die acht Banden der Fraktion zwei der Mutante Mel-3 unterschieden sich in ihrer Auswirkung auf den Oxidative Burst. Hier wirkten sich nur die Fraktionen 1,4,5 hemmend auf die ROS-Synthese aus. Die weiterhin untersuchten Reinsubstanzen Exophialin und Malassezin hatten selbst in hohen Konzentrationen keinen signifikanten Einfluß auf die ROS-Synthese. Insgesamt konnte gezeigt werden, daß Exophiala dermatitidis Wildtyp und seine Mutanten unter den eingesetzten Anzuchtbedingungen ein sehr unterschiedliches Spektrum an pigmentierten Indolderivaten bilden, das sich stets reproduzieren ließ. Es wurde eine Hemmung der ROS-Synthese durch diese Pigmente in Form von Rohextrakten einzelner Stämme, in Form weiter aufgereinigten Säulenchromatographie-Fraktionen, sowie in Form von Einzelbanden beobachtet. Die läßt Rückschlüsse auf deren wichtige Rolle in der Pathogenese von Exophiala dermatitidis-Infektionen bei Mensch und Tier zu. Die aus diesen Resultaten hervorgehenden neuen Fragestellungen sind weiteren Untersuchungen vorbehalten.In 1998, the working group of Prof. Mayser first described some particular indole derivatives in the yeast Malassezia furfur, which might be clinically related to the skin disease pityriasis versicolor. In 2006, Nies et al. were the first ones able to show the production of these indole derivatives in Exophiala dermatitidis. The aim of this study was to compare the pigment spectrum of different species of Exophiala dermatitidis and to clarify whether they are part of the pathogenese of infections with Exophiala dermatitidis. The wildtype and its mutants Mel-1, Mel-3 and Mel-4 were cultivated on minimal agar as a base for pigment production. The pigment was extracted after a period of growth of 4 weeks and chromatographically purified to obtain pure substances. Structural analyses of the isolated substances were performed at Munich Technological University. Following the studies of Schnitzler et al. (1999), unlabeled yeast cells were incubated in heparinized whole blood and dihydrorhodamine 123 was added as an indicator for the ROS-synthesis in the granulocytes. Flow cytometric analysis was performed in a FACScan flow cytometer. By investigating the pigment spectrum of Exophiala dermatitidis, the synthesis of Pityriacitrin, Pityriarubin C, Exophialin and Desoxyexophialin in the wildtype could be shown. In former studies, the pigments of the mutant Mel-1 were already investigated by comparative DC and the indole pyruvate Pityriacitrin was found to be produced by this yeast. In the present study, this achievement could be approved by HPLC and the analysis of Munich Technological University. Exophialin, an almost ankwown indole derivate which was isolated from Mel-3 for the first time by Nies et al. (2006) was found in two violet DC-bands of Mel-1 (Rf=0,38; Rf=0,56). The indole derivates Exophialin, Pityriarubin C and Pityriacitrin were detected in the mutant Mel-3 in this present study. The degree of the oxidative burst evoked by Exophiala dermatitidis wildtype was found to be lower when it was compared with its mutant Mel-3. The wildtype is probably protected from the oxidative burst by its synthesised Melanin. When the raw extracts of the wildtype, Mel-1, Mel-3 and Mel-4 were added in the experimental series of oxidative burst, evoked oxidative burst levels were also found to be lower compared with the contol without any indole derivates. The raw extract of Mel-3 inhibited the oxidative burst most. The sephadex-fractions of the mutant Mel-3 had different influences on the synthesis of ROS. Fractions two and six suppressed the oxidative burst in all time levels between 0 and 60 minutes strongly, while fractions three and eight had no influence on the synthesis of ROS at any time. Fraction six was subdivided by preparative thin layer chromatography into eight bands and the bands 5-8 were able to inhibit evoked oxidative burst levels. Beside the already known Pityriarubin C and other fluorochromes these three bands 5-8 include an unknown greycoloured pigment. This grey pigment was able to inhibit the synthesis of ROS strongly, while Pityriarubin C had no influence on the evoked oxidative burst. The eight bands of fraction two of the mutant Mel-3 differed in the way tey influenzed the oxidative burst. Only fractions 1,4,5 led to a decreased oxidative burst level. Although added in high concentrations, the pure substances Exophialin and Malassezin did not have any influence on the synthesis of ROS. I summary can be said that the wildtype of Exophiala dermatitidis an its mutants are able to produce a very bright and different spectrum of pigmented indole derivates when cultivated on minimal agar. This pigment spectrum is reproducable at any time. An inhibition of the ROS synthesis was shown by these indole derivates when they were added as raw extracts, fractions of thin layer chromatographie and as single bunches. The indole derivates seem to play an important role in the pathogenesis of infections in human beings and animals caused by Exophiala dermatitidis

Absence of renal hypoxia in the subacute phase of severe renal ischemia reperfusion injury

 This is the author accepted manuscript. The final version is available from American Physiological Society via the DOI in this recordTissue hypoxia has been proposed as an important event in renal ischemia reperfusion injury (IRI) particularly during the period of ischemia and in the immediate hours following reperfusion. However, little is known about renal oxygenation during the subacute phase of IRI. We employed four different methods to assess the temporal and spatial changes in tissue oxygenation during the subacute phase (24 h and 5 days after reperfusion) of a severe form of renal IRI in rats. We hypothesized that the kidney is hypoxic 24 h and 5 days after an hour of bilateral renal ischemia, driven by a disturbed balance between renal oxygen delivery (DO2) and oxygen consumption (VO2). Renal DO2 was not significantly reduced in the subacute phase of IRI. In contrast, renal VO2 was 55% less 24 h, and 49% less 5 days after reperfusion than after sham-ischemia. Inner medullary tissue PO2, measured by radiotelemetry was 25 {plus minus} 12% greater 24 h after ischemia than after sham-ischemia. By 5 days after reperfusion, tissue PO2 was similar to that in rats subjected to sham-ischemia. Tissue PO2 measured by Clark electrode was consistently greater 24 h, but not 5 days, after ischemia than after sham-ischemia. Cellular hypoxia, assessed by pimonidazole adduct immunohistochemistry, was largely absent at both time-points and tissue levels of hypoxia inducible factors were downregulated following renal ischemia. Thus, in this model of severe IRI, tissue hypoxia does not appear to be an obligatory event during the subacute phase, likely due to the markedly reduced oxygen consumption.British Heart FoundationBritish Heart FoundationNational Health and Medical Research Council of AustraliaEuropean Union, Seventh Framework Programm

The use of rapid review methods in health technology assessments: 3 case studies.

BACKGROUND: Rapid reviews are of increasing importance within health technology assessment due to time and resource constraints. There are many rapid review methods available although there is little guidance as to the most suitable methods. We present three case studies employing differing methods to suit the evidence base for each review and outline some issues to consider when selecting an appropriate method. METHODS: Three recently completed systematic review short reports produced for the UK National Institute for Health Research were examined. Different approaches to rapid review methods were used in the three reports which were undertaken to inform the commissioning of services within the NHS and to inform future trial design. We describe the methods used, the reasoning behind the choice of methods and explore the strengths and weaknesses of each method. RESULTS: Rapid review methods were chosen to meet the needs of the review and each review had distinctly different challenges such as heterogeneity in terms of populations, interventions, comparators and outcome measures (PICO) and/or large numbers of relevant trials. All reviews included at least 10 randomised controlled trials (RCTs), each with numerous included outcomes. For the first case study (sexual health interventions), very diverse studies in terms of PICO were included. P-values and summary information only were presented due to substantial heterogeneity between studies and outcomes measured. For the second case study (premature ejaculation treatments), there were over 100 RCTs but also several existing systematic reviews. Data for meta-analyses were extracted directly from existing systematic reviews with new RCT data added where available. For the final case study (cannabis cessation therapies), studies included a wide range of interventions and considerable variation in study populations and outcomes. A brief summary of the key findings for each study was presented and narrative synthesis used to summarise results for each pair of interventions compared. CONCLUSIONS: Rapid review methods need to be chosen to meet both the nature of the evidence base of a review and the challenges presented by the included studies. Appropriate methods should be chosen after an assessment of the evidence base

Use of AUDIT-C alcohol screening tool in NHS general dental practices in North London

Background: The numerous health risks of excessive alcohol consumption are well documented. Individuals at risk of harm from alcohol consumption can be identified through alcohol screening tools; however, there is limited research regarding their use in general dental practices. Methods: Data were collected as part of a feasibility trial evaluating delivery of brief alcohol advice in general dental practices in North London. Patient demographics and health-related behaviours were collected, and the Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) tool was used to assess alcohol consumption patterns. Results: The analytical sample comprised 552 dental patients, of whom approximately half (46%) were drinking alcohol at hazardous levels. Males, younger adults, those who consumed red meat weekly and smokers all had significantly increased risks of excessive alcohol consumption. Smokers were more likely to consume excessive levels of alcohol irrespective of smoking frequency. Notable sex differences in alcohol consumption were identified, with males being more likely to consume alcohol frequently and in larger quantities than females. Conclusion: The AUDIT-C tool can be used in general dental practice to screen for harmful levels of alcohol consumption. Clear associations exist between patient demographics, health behaviours and excessive alcohol consumption

Carbohydrate supplementation during prolonged cycling exercise spares muscle glycogen but does not affect intramyocellular lipid use

Using contemporary stable-isotope methodology and fluorescence microscopy, we assessed the impact of carbohydrate supplementation on whole-body and fiber-type-specific intramyocellular triacylglycerol (IMTG) and glycogen use during prolonged endurance exercise. Ten endurance-trained male subjects were studied twice during 3 h of cycling at 63 ± 4% of maximal O2 uptake with either glucose ingestion (CHO trial; 0.7 g CHO kg−1 h−1) or without (CON placebo trial; water only). Continuous infusions with [U-13C] palmitate and [6,6-2H2] glucose were applied to quantify plasma free fatty acids (FFA) and glucose oxidation rates and to estimate intramyocellular lipid and glycogen use. Before and after exercise, muscle biopsy samples were taken to quantify fiber-type-specific IMTG and glycogen content. Plasma glucose rate of appearance (Ra) and carbohydrate oxidation rates were substantially greater in the CHO vs CON trial. Carbohydrate supplementation resulted in a lower muscle glycogen use during the first hour of exercise in the CHO vs CON trial, resulting in a 38 ± 19 and 57 ± 22% decreased utilization in type I and II muscle-fiber glycogen content, respectively. In the CHO trial, both plasma FFA Ra and subsequent plasma FFA concentrations were lower, resulting in a 34 ± 12% reduction in plasma FFA oxidation rates during exercise (P < 0.05). Carbohydrate intake did not augment IMTG utilization, as fluorescence microscopy revealed a 76 ± 21 and 78 ± 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively. We conclude that carbohydrate supplementation during prolonged cycling exercise does not modulate IMTG use but spares muscle glycogen use during the initial stages of exercise in endurance-trained men

Ser649 and Ser650 Are the Major Determinants of Protein Kinase A-Mediated Activation of Human Hormone-Sensitive Lipase against Lipid Substrates

BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate. CONCLUSIONS: Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region

Which circulating antioxidant vitamins are confounded by socioeconomic deprivation? The MIDSPAN family study

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Antioxidant vitamins are often described as having “independent” associations with risk of cancer, cardiovascular disease (CVD) and mortality. We aimed to compare to what extent a range of antioxidant vitamins and carotenoids are associated with adulthood and childhood markers of socioeconomic deprivation and to adverse lifestyle factors.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methods and Findings:&lt;/b&gt; Socioeconomic and lifestyle measures were available in 1040 men and 1298 women from the MIDSPAN Family Study (30–59 years at baseline) together with circulating levels of vitamins A, C, E, and carotenoids (α-carotene, β-carotene, lutein and lycopene). Markers of socioeconomic deprivation in adulthood were consistently as strongly associated with lower vitamin C and carotenoid levels as markers of adverse lifestyle; the inverse association with overcrowding was particularly consistent (vitamin C and carotenoids range from 19.1% [95% CI 30.3–6.0] to 38.8% [49.9–25.3] lower among those in overcrowded residencies). These associations were consistent after adjusting for month, classical CVD risk factors, body mass index, physical activity, vitamin supplements, dietary fat and fibre intake. Similar, but weaker, associations were seen for childhood markers of deprivation. The association of vitamin A or E were strikingly different; several adult adverse lifestyle factors associated with higher levels of vitamin A and E, including high alcohol intake for vitamin A (9.5% [5.7–13.5]) and waist hip ratio for vitamin E (9.5% [4.8–14.4]), with the latter associations partially explained by classical risk factors, particularly cholesterol levels.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Plasma vitamin C and carotenoids have strong inverse associations with adulthood markers of social deprivation, whereas vitamin A and E appear positively related to specific adverse lifestyle factors. These findings should help researchers better contextualize blood antioxidant vitamin levels by illustrating the potential limitations associated with making causal inferences without consideration of social deprivation.&lt;/p&gt