Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

Triglycerides (or triacylglycerols) represent the major form of stored energy in eukaryotes. Triglyceride synthesis has been assumed to occur primarily through acyl CoA:diacylglycerol transferase (Dgat), a microsomal enzyme that catalyses the final and only committed step in the glycerol phosphate pathway. Therefore, Dgat has been considered necessary for adipose tissue formation and essential for survival. Here we show that Dgat-deficient (Dgat-/-) mice are viable and can still synthesize triglycerides. Moreover, these mice are lean and resistant to diet-induced obesity. The obesity resistance involves increased energy expenditure and increased activity. Dgat deficiency also alters triglyceride metabolism in other tissues, including the mammary gland, where lactation is defective in Dgat-/- females. Our findings indicate that multiple mechanisms exist for triglyceride synthesis and suggest that the selective inhibition of Dgat-mediated triglyceride synthesis may be useful for treating obesity.

Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes. We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples. To test the system, 864 physically mapped X clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements. The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores. A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores. The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples. This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies. As a general approach to this problem, we have developed a system for making microarrays of DNA samples on glass substrates, probing them by hybridization with complex fluorescentlabeled probes, and using a laser-scanning microscope to detect the fluorescent signals representing hybridization. Fluorescent labeling allows for simultaneous hybridization and separate detection of the hybridization signal from two or more probes. This in turn allows very accurate and reliable measurement of the relative abundance of specific sequences in two complex samples. with a mixed probe consisting of lissaminelabeled DNA from the 6 largest yeast chromosomes together with fluorescein-labeled DNA from the 10 smallest yeast chromosomes. A red color indicates that yeast sequences present in the lissamine-labeled hybridization probe hybridized to an array element. A yellow-green color indicates that yeast sequences present in the fluorescein-labeled hybridization probe hybridized to an array element. An orange color indicates cross-hybridization of both chromosome pools to an array element (e.g., dispersed repetitive elements, such as Tyl elements). RESULTS Array Hybridization PatternEach clone was spotted twice, resulting in duplicate hybridization patterns in adjacent quadrants of the array. Control DNA spots, which were randomly amplified in the same manner as the X clone array elements, are located in the bottom corner of each quadrant. "A" points to a pair of spots containing total yeast genomic DNA. These spots appear orange because both chromosome pools hybridized to yeast genomic DNA. The negative controls are as follows: "B" points to a pair of spots of wild-type X DNA, "C" points to a pair of human genomic DNA spots, and "D" points to a pair of ~X174 DNA spots. The lack of a hybridization signal at these three negative control spots indicates that the hybridization was specific for yeast sequences.

Raltegravir (RAL) and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) efficiently block viral replication in vitroand suppress viremia in patients. These small molecules bind to the IN active site, causing it to disengage from the deoxyadenosine at the 3Ј end of viral DNA. The emergence of viral strains that are highly resistant to RAL underscores the pressing need to develop INSTIs with improved resistance profiles. Herein, we show that the candidate second-generation drug dolutegravir (DTG, S/GSK1349572) effectively inhibits a panel of HIV-1 IN variants resistant to first-generation INSTIs. To elucidate the structural basis for the increased potency of DTG against RAL-resistant INs, we determined crystal structures of wild-type and mutant prototype foamy virus intasomes bound to this compound. The overall IN binding mode of DTG is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048. Both second-generation INSTIs occupy almost the same physical space within the IN active site and make contacts with the ␤4 -␣2 loop of the catalytic core domain. The extended linker region connecting the metal chelating core and the halobenzyl group of DTG allows it to enter farther into the pocket vacated by the displaced viral DNA base and to make more intimate contacts with viral DNA, compared with those made by RAL and other INSTIs. In addition, our structures suggest that DTG has the ability to subtly readjust its position and conformation in response to structural changes in the active sites of RAL-resistant INs.

Abstract. Understanding and quantifying the global methane (CH4) budget is important for assessing realistic pathways to mitigate climate change. Atmospheric emissions and concentrations of CH4 are continuing to increase, making CH4 the second most important human-influenced greenhouse gas in terms of climate forcing, after carbon dioxide (CO2). Assessing the relative importance of CH4 in comparison to CO2 is complicated by its shorter atmospheric lifetime, stronger warming potential, and atmospheric growth rate variations over the past decade, the causes of which are still debated. Two major difficulties in reducing uncertainties arise from the variety of geographically overlapping CH4 sources and from the destruction of CH4 by short-lived hydroxyl radicals (OH). To address these difficulties, we have established a consortium of multi-disciplinary scientists under the umbrella of the Global Carbon Project to synthesize and stimulate new research aimed at improving and regularly updating the global methane budget. Following Saunois et al. (2016), we present here the second version of the living review paper dedicated to the decadal methane budget, integrating results of top-down studies (atmospheric observations within an atmospheric inverse-modelling framework) and bottom-up estimates (including process-based models for estimating land surface emissions and atmospheric chemistry, inventories of anthropogenic emissions, and data-driven extrapolations). For the 2008–2017 decade, global methane emissions are estimated by atmospheric inversions (top-down approach) to be 572 Tg CH4 yr−1 (range 538–593, corresponding to the minimum and maximum estimates of the ensemble), of which 357 Tg CH4 yr−1 or ~ 60 % are attributed to anthropogenic sources (range 50–65 %). This total emission is 27 Tg CH4 yr−1 larger than the value estimated for the period 2000–2009 and 24 Tg CH4 yr−1 larger than the one reported in the previous budget for the period 2003–2012 (Saunois et al. 2016). Since 2012, global CH4 emissions have been tracking the carbon intensive scenarios developed by the Intergovernmental Panel on Climate Change (Gidden et al., 2019). Bottom-up methods suggest larger global emissions (737 Tg CH4 yr−1, range 583–880) than top-down inversion methods, mostly because of larger estimated natural emissions from sources such as natural wetlands, other inland water systems, and geological sources. However the strength of the atmospheric constraints on the top-down budget, suggest that these bottom-up emissions are overestimated. The latitudinal distribution of atmospheric-based emissions indicates a predominance of tropical emissions (~ 65 % of the global budget,

IntroductionBecause obesity results from an imbalance between energy input and output, with most of the excess calories stored as triglycerides (or triacylglycerols), inhibition of triglyceride synthesis may prevent or reverse obesity (1). One of the key enzymes in triglyceride synthesis is acyl coenzyme A:diacylglycerol acyltransferase (acyl CoA:diacylglycerol acyltransferase, or DGAT), which catalyzes the final step in mammalian triglyceride synthesis. Two DGAT enzymes (DGAT1 and DGAT2) have been identified (2, 3). DGAT1 activity is widely distributed, and its gene (Dgat1) is expressed in all tissues examined (2). To investigate the effects of disrupting triglyceride synthesis on energy and glucose metabolism, we generated DGAT1-deficient (Dgat1 -/-) mice (4). Dgat1 -/-mice have triglycerides in their adipose tissue and normal plasma triglyceride levels. The residual triglyceride synthesis presumably occurs through the actions of DGAT2 and perhaps additional mechanisms of triglyceride synthesis (5). Dgat1 -/-mice are resistant to diet-induced obesity because of increased energy expenditure. This increase is partially mediated by a twofold increase in physical activity in Dgat1 -/-mice fed a high-fat diet. These mice also tend to have enhanced glucose disposal after a glucose load on either a chow or a high-fat diet (4).How does DGAT1 deficiency affect energy and glucose metabolism? One plausible mechanism is by modulating tissue triglyceride metabolism. Increased triglyceride content in tissues such as skeletal muscle and liver correlates with insulin resistance (6-8). Moreover, increased adiposity is associated with resistance to leptin, an adipocyte-derived hormone that enhances energy expenditure and insulin sensitivity (9, 10). Because DGAT1 deficiency in mice is not associated with a compensatory increase in DGAT2 mRNA expression (3), we hypothesized that Dgat1 -/-mice have reduced levels of tissue triglycerides and that these reductions are associated with increased sensitivity to insulin and to leptin. To test this hypothesis, we measured tissue triglyceride levels in Dgat1 -/-mice, and we performed hyperinsulinemic-euglycemic clamp and leptin infusion studies. We also studied the effects of DGAT1 deficiency on energy and glucose metabolism in agouti yellow (A Y /a) and leptindeficient (ob/ob) mice, two genetic models of obesity and insulin resistance. Our findings provide new insights into how alterations in triglyceride synthesis affect insulin and leptin sensitivity. MethodsMice. Dgat1 -/-mice (∼95% C57BL/6 and 5% 129/SvJae background) were generated previously (4). Wild-type (Dgat1 +/+ ), ob/+, and A Y /a mice (all in C57BL/6 background) were from the The Jackson Laboratory (Bar Harbor, Maine, USA). A Y /a mice are obese and insulin resistant, reflecting the antagonism of melanocyte- Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in mammalian triglyceride synthesis. DGAT1-deficient mice are resistant to diet-induced obesity thro...

Dietary triacylglycerols are a major source of energy for animals. The absorption of dietary triacylglycerols involves their hydrolysis to free fatty acids and monoacylglycerols in the intestinal lumen, the uptake of these products into enterocytes, the resynthesis of triacylgylcerols, and the incorporation of newly synthesized triacylglycerols into nascent chylomicrons for secretion. In enterocytes, the final step in triacylglycerol synthesis is believed to be catalyzed primarily through the actions of acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. In this study, we analyzed intestinal triacylglycerol absorption and chylomicron synthesis and secretion in DGAT1-deficient (Dgat1 ؊/؊ ) mice. Surprisingly, DGAT1 was not essential for quantitative dietary triacylglycerol absorption, even in mice fed a high fat diet, or for the synthesis of chylomicrons. However, Dgat1 ؊/؊ mice had reduced postabsorptive chylomicronemia (1 h after a high fat challenge) and accumulated neutrallipid droplets in the cytoplasm of enterocytes when chronically fed a high fat diet. These results suggest a reduced rate of triacylglycerol absorption in Dgat1 mice. Analysis of intestine from Dgat1؊/؊ mice revealed activity for two other enzymes, DGAT2 and diacylglycerol transacylase, that catalyze triacylglycerol synthesis and apparently help to compensate for the absence of DGAT1. Our findings indicate that multiple mechanisms for triacylglycerol synthesis in the intestine facilitate triacylglycerol absorption.The absorption of triacylglycerols by the intestine is highly efficient, and more than 95% of dietary triacylglycerols is absorbed, even if the diet is rich in fat. By comparison, only 30 -70% of dietary cholesterol is absorbed in most animals (1). The high efficiency of triacylglycerol absorption is likely due to an evolutionary pressure that maximized the ability to absorb rich sources of energy (such as fat) when food sources were scarce.Intestinal triacylglycerol absorption occurs by a series of steps in which dietary triacylglycerols are first hydrolyzed in the intestinal lumen and then resynthesized within enterocytes. In the lumen, dietary triacylglycerols are hydrolyzed by lipases to generate free fatty acids and monoacylglycerols. These molecules are taken up by enterocytes and then enter the triacylglycerol biosynthesis pathways. The triacylglycerol products are incorporated into nascent chylomicrons, which are subsequently secreted from enterocytes and enter the lymphatic system.Triacylglycerol biosynthesis in the intestine is believed to occur mainly through the monoacylglycerol pathway. In this pathway, monoacylglycerol and fatty acyl-CoA are covalently joined to form diacylglycerol in a reaction catalyzed by monoacylglycerol acyltransferase (MGAT) 1 (2). Diacylglycerol and fatty acyl-CoA are then used to synthesize triacylglycerol in a reaction catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. High levels of DGAT activity are present in the small intestine (3-5), and both known DGAT genes, Dgat1...

No abstract