Targeting Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) with small molecule inhibitors for the treatment of metabolic diseases

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in triglyceride synthesis. Findings from genetically modified mice as well as pharmacological studies suggest that inhibition of DGAT1 is a promising strategy for the treatment of obesity and type 2 diabetes. Here we characterize a tool DGAT1 inhibitor compound, T863. We found that T863 is a potent inhibitor for both human and mouse DGAT1 in vitro, which acts on the acyl-CoA binding site of DGAT1 and inhibits DGAT1-mediated triacylglycerol formation in cells. In an acute lipid challenge model, oral administration of T863 significantly delayed fat absorption and resulted in lipid accumulation in the distal small intestine of mice, mimicking the effects of genetic ablation of DGAT1. In diet-induced obese mice, oral administration of T863 for 2 weeks caused weight loss, reduction in serum and liver triglycerides, and improved insulin sensitivity. In addition to the expected triglyceride-lowering activity, T863 also lowered serum cholesterol. Hepatic IRS2 protein was dramatically up-regulated in mice treated with T863, possibly contributing to improved insulin sensitivity. In differentiated 3T3-L1 adipocytes, T863 enhanced insulin-stimulated glucose uptake, suggesting a possible role for adipocytes to improve insulin sensitivity upon DGAT1 inhibition. These results reveal novel mechanistic insights into the insulin-sensitizing effects of DGAT1 inhibition in mouse models. Taken together, our study provides a comprehensive evaluation of a small molecule inhibitor for DGAT1 and suggests that pharmacological inhibition of DGAT1 holds promise in treating diverse metabolic disorders.     

Metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin

Polyglycerophospholipids (PGPLs) such as bismonoacylglycerophosphate (BMP) and cardiolipin are important membrane phospholipid species for the maintenance of membrane integrity. While BMP serves as membrane curvature regulator in multivesicular bodies for efficient lysosomal enzyme function, cardiolipin stabilizes the electron transfer complex in the inner mitochondrial membrane, which is crucial for physiological ATP production. Beside their membrane modulatory functions PGPLs play an important role in various signaling events. Although a number of disease associations were found for PGPL species, detailed information about their molecular role still remains unknown. This article reviews the known biological functions of PGPLs and the existing mass spectrometric methods. We discuss the different analytical strategies and how ESI–MS/MS can expand our understanding of PGPL homeostasis.     

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 overexpression enhances postprandial triglyceridemic response and exacerbates high fat diet-induced hepatic triacylglycerol storage

Intestinal acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) is important in the cellular and physiological responses to dietary fat. To determine the effect of increased intestinal DGAT2 on cellular and physiological responses to acute and chronic dietary fat challenges, we generated mice with intestine-specific overexpression of DGAT2 and compared them with intestine-specific overexpression of DGAT1 and wild-type (WT) mice. We found that when intestinal DGAT2 is present in excess, triacylglycerol (TG) secretion from enterocytes is enhanced compared to WT mice; however, TG storage within enterocytes is similar compared to WT mice. We found that when intestinal DGAT2 is present in excess, mRNA levels of genes involved in fatty acid oxidation were reduced. This result suggests that reduced fatty acid oxidation may contribute to increased TG secretion by overexpression of DGAT2 in intestine. Furthermore, this enhanced supply of TG for secretion in Dgat2^Int mice may be a significant contributing factor to the elevated fasting plasma TG and exacerbated hepatic TG storage in response to a chronic HFD. These results highlight that altering fatty acid and TG metabolism within enterocytes has the capacity to alter systemic delivery of dietary fat and may serve as an effective target for preventing and treating metabolic diseases such as hepatic steatosis.     

Retinol and retinyl esters: biochemistry and physiology: Thematic Review Series: Fat-Soluble Vitamins: Vitamin A

By definition, a vitamin is a substance that must be obtained regularly from the diet. Vitamin A must be acquired from the diet, but unlike most vitamins, it can also be stored within the body in relatively high levels. For humans living in developed nations or animals living in present-day vivariums, stored vitamin A concentrations can become relatively high, reaching levels that can protect against the adverse effects of insufficient vitamin A dietary intake for six months, or even much longer. The ability to accumulate vitamin A stores lessens the need for routinely consuming vitamin A in the diet, and this provides a selective advantage to the organism. The molecular processes that underlie this selective advantage include efficient mechanisms to acquire vitamin A from the diet, efficient and overlapping mechanisms for the transport of vitamin A in the circulation, a specific mechanism allowing for vitamin A storage, and a mechanism for mobilizing vitamin A from these stores in response to tissue needs. These processes are considered in this review.     

Diacylglycerol acyltransferase-2 contains a c-terminal sequence that interacts with lipid droplets

Diacylglycerol acyltranferase-2 (DGAT2) is a resident protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerol. When lipid droplet formation is stimulated by incubating cells with fatty acids, DGAT2 becomes concentrated around the surface of cytosolic lipid droplets. Using confocal microscopy and directed mutagenesis, we have identified a 17-amino acid sequence in the C-terminal region of DGAT2 that is necessary and sufficient for targeting DGAT2 to lipid droplets. When this region was deleted, DGAT2 remained in the ER and did not target to lipid droplets. Fusing this sequence to mCherry directed the fluorescent reporter to lipid droplets. Similarly, when the corresponding region of monoacylglycerol acyltransferase-2 (MGAT2) was replaced with this sequence, MGAT2 was also targeted to lipid droplets. Lastly, we demonstrated that DGAT2 in ER membranes is continuous with lipid droplets. We propose a new model whereby DGAT2 remains in the ER during lipid droplet formation via it's transmembrane domains and interacts with nascent lipid droplets via its C-terminal lipid droplet interacting domain as they expand.     

Real time quantitative analysis of lipid storage and lipolysis pathways by confocal spectral imaging of intracellular micropolarity

Organisms store fatty acids in triacylglycerols in the form of lipid droplets, or hydrolyze triacylglycerols in response to energetic demands via activation of lipolytic or storage pathways. These pathways are complex sets of sequential reactions that are finely regulated in different cell types. Here we present a high spatial and temporal resolution-based method for the quantification of the turnover of fatty acids into triglycerides in live cells without introducing sample preparation artifacts.We performed confocal spectral imaging of intracellular micropolarity in cultured insulin secreting beta cells to detect micropolarity variations as they occur in time and at different pixels of microscope images. Acquired data are then analyzed in the framework of the spectral phasors technique.The method furnishes a metabolic parameter, which quantitatively assesses fatty acids - triacylglycerols turnover and the activation of lipolysis and storage pathways. Moreover, it provides a polarity profile, which represents the contribution of hyperpolar, polar and non-polar classes of lipids. These three different classes can be visualized on the image at a submicrometer resolution, revealing the spatial localization of lipids in cells under physiological and pathological settings.This new method allows for a fine-tuned, real-time visualization of the turnover of fatty acids into triglycerides in live cells with submicrometric resolution. It also detects imbalances between lipid storage and usage, which may lead to metabolic disorders within living cells and organisms.     

Discovery of dimethyl pent-4-ynoic acid derivatives,as potent and orally bioavailable DGAT1 inhibitors that suppress body weight in diet-induced mouse obesity model

Diacylglycerol acyltransferase (DGAT) is expressed abundantly in intestine, liver, and adipose tissues. DGAT1 is the crucial and rate-limiting enzyme that mediates the final step in triacylglycerol (TAG) resynthesis during dietary fat absorption. However, too much triacylglycerol (TAG) reserve will lead to genetic obesity (Hubert et al., 2000). DGAT1 knockout mice could survive and displayed a reduction in the postprandial rise of plasma TG, and increased sensitivity of insulin and leptin. Here we report the discovery and characterization of a novel selective DGAT1 inhibitor 29 to potentially treat obesity. Compound 29 showed lipid lowering effect in mouse lipid tolerance test (LTT) and also reduced body weight in DIO mice without observable liver damage.     

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates

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