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Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research., Given the recent growing interest in interorganelle membrane contact sites, the field will benefit from clear rules to define and study them. In this Perspective, a panel of experts aims to provide this growing field with guidelines for experimental definition and analysis.

Hydroxybutyrylcarnitine (HB-carnitine) is a metabolite that has been associated with insulin resistance and type 2 diabetes mellitus. It is currently unknown whether HB-carnitine can be produced from D-3-hydroxybutyrate (D-3HB), a ketone body; but its formation from L-3-HB-CoA, a fatty acid beta-oxidation intermediate, is well established. We aimed to assess which stereoisomers of 3-HB-carnitine are present in vivo. Ketosis and increased fatty acid oxidation were induced in 12 lean healthy men by a 38-hour fasting period. The D-3HB kinetics (stable isotope technique) and stereoisomers of muscle 3-HB-carnitine (high-performance liquid chromatography/ultra-performance liquid chromatography tandem mass spectrometry) were measured. Muscle D-3HB-carnitine content was much higher compared with L-3HB-carnitine. In addition, muscle D-3HB-carnitine correlated significantly with D-3-HB production. Following the finding that a ketone body can be converted into a carnitine ester in vivo, we show in vitro that D-3-HB can be converted into HB-carnitine (ketocarnitine) via an acyl-CoA synthetase reaction in several tissues including human muscle. During fasting, HB-carnitine in muscle is derived mainly from the ketone body D-3HB. The role of D-3HB-carnitine synthesis in metabolism remains to be elucidated. (c) 2012 Elsevier Inc. All rights reserved

Peripheral blood mononuclear cells (PBMC) are easily obtainable cells from blood whose gene expression profiles have been proven to be highly robust in distinguishing a disease state from healthy state. Sterol metabolism is of physiological importance, and although its nutritional response in liver has been described, it is poorly studied in PBMC. To examine if PBMC sterol metabolism reflects diet-induced physiological responses, we analysed the whole genome gene expression response of PBMC and focused on sterol metabolism-related genes affected by different feeding conditions (ad libitum feeding, fasting, and refeeding) in normoweight (control) rats and in diet-induced (cafeteria) obese rats. Our results of microarray analysis show that, in control rats, 21 genes involved in sterol metabolism were regulated by the different feeding conditions, whereas in cafeteria-obese rats, only seven genes showed a changed expression. Most of the genes identified were classified into three pathways: sterol biosynthesis, cholesterol transport and uptake and sterol signaling. The expression profile of these genes was similar to that previously described for liver, decreasing in response to fasting conditions and recovering the levels found in fed animals after 6-h-refeeding. In addition, our data and the comparable expression pattern of sterol metabolism-related genes between PBMC and liver suggest similar sterol regulatory element-binding protein-mediated regulatory mechanisms in response to feeding conditions in both tissues. In conclusion, the expression of genes involved in sterol metabolism is highly controlled by feeding conditions in PBMC of control rats, but this control is impaired in cafeteria-obese animals. The pathophysiological significance of this impairment requires further investigation.

The transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha) is an important regulator of hepatic lipid metabolism. While PPARalpha is known to activate transcription of numerous genes, no comprehensive picture of PPARalpha binding to endogenous genes has yet been reported. To fill this gap, we performed Chromatin immunoprecipitation (ChIP)-chip in combination with transcriptional profiling on HepG2 human hepatoma cells treated with the PPARalpha agonist GW7647. We found that GW7647 increased PPARalpha binding to 4220 binding regions. GW7647-induced binding regions showed a bias around the transcription start site and most contained a predicted PPAR binding motif. Several genes known to be regulated by PPARalpha, such as ACOX1, SULT2A1, ACADL, CD36, IGFBP1 and G0S2, showed GW7647-induced PPARalpha binding to their promoter. A GW7647-induced PPARalpha-binding region was also assigned to SREBP-targets HMGCS1, HMGCR, FDFT1, SC4MOL, and LPIN1, expression of which was induced by GW7647, suggesting cross-talk between PPARalpha and SREBP signaling. Our data furthermore demonstrate interaction between PPARalpha and STAT transcription factors in PPARalpha-mediated transcriptional repression, and suggest interaction between PPARalpha and TBP, and PPARalpha and C/EBPalpha in PPARalpha-mediated transcriptional activation. Overall, our analysis leads to important new insights into the mechanisms and impact of transcriptional regulation by PPARalpha in human liver and highlight the importance of cross-talk with other transcription factors.

Summary The mitochondrial aspartate–glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate– aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.

Little is known about endogenous surfactant metabolism in infants, because radioactive isotopes used for this purpose in animals cannot be used in humans. We developed a novel and safe method to measure the endogenous surfactant kinetics in vivo in humans by using stable isotope labeled fatty acids. We infused albumin-bound [U-C-13]palmitic acid (PA) and [U-C-13]linoleic acid (LLA) for 24 h in eight critically ill infants (mean +/- SD; weight. 3.7 +/- 1.3 kg; age: 51.3 +/- 61.6 d) who required mechanical ventilation, The C-13 enrichment of PA and LLA in surfactant phosphatidylcholine (PC), obtained from tracheal aspirates, was measured by gas chromatography combustion interface-isotope ratio mass spectrometry. We measured a significant incorporation of both C-13-PA and C-13-LLA into surfactant PC. PC-PA and PC-LW became enriched after 8.7 +/- 4.9 h (range: 3.4-17.3) and 10.0 +/- 7.2 h (range: 3.0-22.4), respectively; the times at maximum enrichment were 49.2 +/- 8.9 and 45.6 +/- 19.3 h, respectively. The fractional synthesis rate of surfactant PC-PA ranged from 0.4 to 3.4% per h, whereas the fractional synthesis rate of PC-LLA ranged from 0.5 to 3.8% per h. The surfactant PC-PA and PC-LLA half-lives ranged from 16.8 to 177.7 and 23.8 to 144.4 h, respectively. This method provides new data on surfactant metabolism in infants requiring mechanical ventilation. We found that synthesis of surfactant from plasma PA and LLA is a slow process and that there were marked differences in PC kinetics among infants. This variability could be related to differences in lung disease and could affect the clinical course of the respiratory failure.