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Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase
Abstract
Acyl-CoA:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of physiologically important lipids such as triacylglycerol and phospholipids. In the intestine, MGAT plays a major role in the absorption of dietary fat because resynthesis of triacylglycerol is required for the assembly of lipoproteins that transport absorbed fat to other tissues. MGAT activity has also been reported in mammalian liver and white adipose tissue. However, MGAT has never been purified to homogeneity from mammalian tissues, and its gene has not been cloned. We identified a gene that encodes an MGAT (MGAT1) in mice. This gene has sequence homology with members of a recently identified diacylglycerol acyltransferase gene family. Expression of the MGAT1 cDNA in insect cells markedly increased MGAT activity in cell membranes. In addition, MGAT activity was proportional to the level of MGAT1 protein expressed, and the amount of diacylglycerol produced depended on the concentration of either of its substrates, oleoyl-CoA or monooleoylglycerol. In mice, MGAT1 expression and MGAT activity were detected in the stomach, kidney, white and brown adipose tissue, and liver. However, MGAT1 was not expressed in the small intestine, implying the existence of a second MGAT gene. The identification of the MGAT1 gene should greatly facilitate research on the identification of the intestinal MGAT gene and on the function of MGAT enzymes in mammalian glycerolipid metabolism.
... These enzymes also exhibit DGAT activity towards 1,2-DAG and 1,3-DAG, catalyzing two steps in the biosynthesis of TAG from monoacylglycerol [194]. The highest activity of these enzymes is observed in the intestine, but lower activity is also present in the stomach, kidney, adipose tissue, and liver [195]. The main role of these enzymes is the absorption of fatty acids in the small intestine. ...
... The main role of these enzymes is the absorption of fatty acids in the small intestine. There are three isoforms of MOGAT: MOGAT1 [195], MOGAT2 [196], and MOGAT3 [197]. ...
... The expression of MOGAT in the brain is very low [195]. Similarly, in glioblastoma tumors, the expression of MOGAT is also very low [48]. ...
One area of glioblastoma research is the metabolism of tumor cells and detecting differences between tumor and healthy brain tissue metabolism. Here, we review differences in fatty acid metabolism, with a particular focus on the biosynthesis of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) by fatty acid synthase (FASN), elongases, and desaturases. We also describe the significance of individual fatty acids in glioblastoma tumorigenesis, as well as the importance of glycerophospholipid and triacylglycerol synthesis in this process. Specifically, we show the significance and function of various isoforms of glycerol-3-phosphate acyltransferases (GPAT), 1-acylglycerol-3-phosphate O-acyltransferases (AGPAT), lipins, as well as enzymes involved in the synthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and cardiolipin (CL). This review also highlights the involvement of diacylglycerol O-acyltransferase (DGAT) in triacylglycerol biosynthesis. Due to significant gaps in knowledge, the GEPIA database was utilized to demonstrate the significance of individual enzymes in glioblastoma tumorigenesis. Finally, we also describe the significance of lipid droplets in glioblastoma and the impact of fatty acid synthesis, particularly docosahexaenoic acid (DHA), on cell membrane fluidity and signal transduction from the epidermal growth factor receptor (EGFR).
... In addition to the small intestine, the MG pathway is also active in adipose tissue and liver [6][7][8]. There are two identified MGATs (MGAT1 and MGAT2) in mice [9,10], as well as three MGATs (MGAT1, MGAT2, MGAT3) in humans [11][12][13]. Two identified DGATs, DGAT1 and DGAT2, function as the rate-limiting enzymes for TG biosynthesis [14,15]. ...
... TMEM68 is an ER-anchored transmembrane protein and does not localize to LDs [25], which resembles the localization of MGAT1-3 enzymes [9,11,13,33]. TMEM68 also displays some DGAT activity in vitro in addition to MGAT activity similar to MGAT1-3 [34,35]. TMEM68 overexpression increased not only cellular TG levels but also DG levels, further supporting a dual MGAT/DGAT-like function of TMEM68. ...
... Hence, the catalytic and cellular localization properties of TMEM68 closely resemble those of established MGAT enzymes. TMEM68 is highly expressed in mouse brain [25], a tissue that does not express detectable levels of MGAT1 or MGAT2, but exhibits measurable MGAT activity [9,10]. In addition to DG and TG, FFA levels were also increased by TMEM68 overexpression, whereas acylcarnitine level was decreased, which suggested that more FFA was channeled to TG synthesis and less FFA to be oxidized for energy. ...
Triacylglycerol (TG) biosynthesis is an important metabolic process for intracellular storage of surplus energy, intestinal dietary fat absorption, attenuation of lipotoxicity, lipid transportation, lactation and signal transduction in mammals. Transmembrane protein 68 (TMEM68) is an endoplasmic reticulum (ER)-anchored acyltransferase family member of unknown function. In the current study we show that overexpression of TMEM68 promotes TG accumulation and lipid droplet (LD) formation in a conserved active sites-dependent manner. Quantitative targeted lipidomic analysis showed that diacylglycerol (DG), free fatty acid (FFA) and TG levels were increased by TMEM68 expression. In addition, TMEM68 overexpression affected the levels of several glycerophospholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, as well as sterol ester contents. TMEM68 exhibited monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) activities dependent on the conserved active sites in an in vitro assay. The expression of lipogenesis genes, including DGATs, fatty acid synthesis-related genes and peroxisome proliferator-activated receptor γ was upregulated in TMEM68-overexpressing cells. These results together demonstrate for the first time that TMEM68 functions as an acyltransferase and affects lipogenic gene expression, glycerolipid metabolism and TG storage in mammalian cells.
... These TG are then stored in cytosolic lipid droplets or secreted into the circulation as a component of TG-rich chylomicrons via the lymph 18 . Three MGAT isoforms (MGAT1, MGAT2 and MGAT3) have been identified based on their extensive homology to DGAT2 [19][20][21][22] . Each isoform is encoded by a distinct MOGAT gene (MOGAT1, MOGAT2 and MOGAT3) which are expressed in a species-specific manner 23,24 . ...
... Consequently, there is limited information available regarding the contribution of the MGAT pathway to hepatic lipid metabolism. More recent experiments have demonstrated that human liver in fact has robust MGAT activity, although it is not as high as in the small intestine 19,20,23 . Interestingly, all three MOGAT genes are expressed in human liver, while only Mogat1 and Mogat2 are expressed in mouse liver 21,23 . ...
... MGAT3 shares more sequence similarity with DGAT2, than to either MGAT1 or MGAT2 21,22 . MGAT3 also possesses both MGAT and DGAT activities suggesting that it could function as a TG synthase 19,20,[22][23][24] . Its in vivo function has not been determined as Mogat3 in mice is a pseudogene and would not encode a functional protein 24 . ...
The monoacylglycerol acyltransferase (MGAT) pathway has a well-established role in the small intestine where it facilitates the absorption of dietary fat. In enterocytes, MGAT participates in the resynthesis of triacylglycerol using substrates (monoacylglycerol and fatty acids) generated in the gut lumen from the breakdown of triacylglycerol consumed in the diet. MGAT activity is also present in the liver, but its role in triacylglycerol metabolism in this tissue remains unclear. The predominant MGAT isoforms present in human liver appear to be MGAT2 and MGAT3. The objective of this study was to use selective small molecule inhibitors of MGAT2 and MGAT3 to determine the contributions of these enzymes to triacylglycerol production in liver cells. We found that pharmacological inhibition of either enzyme had no effect on TG mass in HepG2 cells but did alter lipid droplet size and number. Inhibition of MGAT2 did result in decreased DG and TG synthesis and TG secretion. Interestingly, MGAT2 preferentially utilized 2-monoacylglycerol derived from free glycerol and not from exogenously added 2-monoacylglycerol. In contrast, inhibition of MGAT3 had very little effect on TG metabolism in HepG2 cells. Additionally, we demonstrated that the MGAT activity of DGAT1 only makes a minor contribution to TG synthesis in intact HepG2 cells. Our data demonstrated that the MGAT pathway has a role in hepatic lipid metabolism with MGAT2, more so than MGAT3, contributing to TG synthesis and secretion.
... These pathways converge at diacylglycerol (DAG); the sole precursor of TAG [7][8][9]. MGATs acylate monoacylglycerol to form DAG and this enzymatic activity is encoded by several genes including Mogat1, Mogat2, and Dgat1 in mice [10][11][12][13]. ...
... Similar to constitutive Mogat1 liver-specific knockout, acute deletion of hepatic Mogat1 did not alter liver TAG content, and actually, slightly increased MGAT activity ( Figure 2E,F). Gene expression of similar acyltransferases (Mogat2, Dgat1, Dgat2) were unaffected by Mogat1 knockout ( Figure 2G) [10][11][12][13]. These data indicate that genetic deletion of Mogat1 in liver of diet-induced obese mice for 3 weeks does not affect glucose or insulin tolerance, and may suggest compensation from other enzymes with MGAT activity [11,12,27]. ...
... Gene expression of similar acyltransferases (Mogat2, Dgat1, Dgat2) were unaffected by Mogat1 knockout ( Figure 2G) [10][11][12][13]. These data indicate that genetic deletion of Mogat1 in liver of diet-induced obese mice for 3 weeks does not affect glucose or insulin tolerance, and may suggest compensation from other enzymes with MGAT activity [11,12,27]. ...
Objective:
Monoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of Mogat1 mRNA, which encodes MGAT1, reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high fat diet (HFD) fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway.
Methods:
Mice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1-knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on a high fat diet and glucose metabolism and insulin sensitivity was assessed after 16 weeks on diet. In some experiments, mice were treated with control scramble or Mogat1 ASOs in the presence or absence of IFNAR-1 neutralizing antibody.
Results:
Genetic deletion of hepatic Mogat1, either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced obesity, insulin sensitivity, and glucose intolerance on a HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression in hepatocytes increased liver MGAT activity and TAG content in low-fat fed mice, but did not cause insulin resistance. Interestingly, multiple Mogat1 ASO sequences improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off target effect. Hepatic IFNAR-1 signaling was activated by multiple Mogat1 ASOs but its blockade did not prevent the effects of either Mogat1 ASO on glucose homeostasis.
Conclusion:
These results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that multiple Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling.
... MGATs were originally identified based on their sequence homology with DGAT2 (Yen et al., 2002;Cheng et al., 2003;Yen and Farese, 2003). They are primary enzymes for the esterification of MAG into DAG, most notably for the absorption of dietary MAG in intestinal epithelial cells. ...
... The DAG product of MGATs can then be fed to DGATs for further esterification into TAG, thus completing the monoacylglycerol pathway. Heterologous expression of MGATs in cultured cells indicated that they are primarily localized to the ER (Yen et al., 2002;Cheng et al., 2003;Yen and Farese, 2003), which enables their functional coupling with DGATs (Jin et al., 2014). In addition, MGAT1 has been reported to localize to LDs (Lee and Kim, 2017). ...
... In addition, MGAT1 has been reported to localize to LDs (Lee and Kim, 2017). MGAT1 and MGAT2 showed tissue restricted expression in humans and mice (Yen et al., 2002;Yen and Farese, 2003). Interestingly, a third MGAT, MGAT3, is expressed in the intestine of humans but not mice (Cheng et al., 2003;Yue et al., 2011). ...
The endoplasmic reticulum (ER) is a hub that coordinates neutral lipid synthesis, storage, and export. To fulfill this role, the ER maintains close contact with lipid droplets (LDs), which are evolutionarily conserved organelles for the storage of neutral lipids. Decades of biochemical evidence points to fatty acid modification and neutral lipid synthesis in the ER. Conceptually, lipid export into extracellular space or lipid retention intracellularly require the subsequent remodeling of an ER membrane leaflet that faces the lumen or cytoplasm, respectively. This is because LDs and very-low-density lipoprotein particles are all structures surrounded by a phospholipid monolayer. While the export of neutral lipids via very-low-density lipoprotein production is well characterized, there has been increasing interest in the mechanisms that underlie neutral lipid retention in LDs. Structural determination, in vitro reconstitution, and localization of key proteins by advanced microscopy techniques collectively enrich models of ER-LD engagement. In this review, we consider current concepts on how LDs emerge from the ER in a directional manner and how sustained ER-LD contacts support LD expansion.
... These pathways converge at diacylglycerol (DAG), which is the sole precursor of TAG [7][8][9]. MGATs acylate monoacylglycerol to form DAG and this enzymatic activity is encoded by several genes including Mogat1, Mogat2, and Dgat1 in mice [10][11][12][13]. ...
... Mogat1 floxed mice were crossed with mice expressing the Cre recombinase transgene under the control of the albumin promoter (Jackson Laboratory, B6.Cg-Speer6-ps1Tg (Alb-cre) 21Mgn/J). Acute liver-specific knockout mice were generated by retro-orbital injection of Mogat1 floxed mice with 2.0 x 10 11 [20]; pooled (2.0 x 10 11 GC total) before injection (Vector Biolabs). For hepatic Mogat1 overexpression, eight-week-old male C57BL6/J mice were given LFD or HFD for six weeks, then administered AAV8-TBG-eGFP or AAV8-TBG-mouse-Mogat1 by retro-orbital injection (Vector Biolabs, VB1743, custom refseq# BC106135). ...
... Similar to constitutive Mogat1 liver-specific knockout, acute deletion of hepatic Mogat1 did not alter liver weight, TAG content, and actually, slightly increased MGAT activity ( Figure 2E-G). Together, these data indicate that genetic deletion of Mogat1 in liver of diet-induced obese mice does not affect glucose or insulin tolerance, and suggests compensation from other enzymes with MGAT activity [11,12,25]. ...
Objective
Monoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of the gene encoding MGAT1, Mogat1 , reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high fat diet (HFD) fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway.
Methods
Mice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1 -knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on a high fat diet and glucose metabolism and insulin sensitivity was assessed after 16 weeks on diet. In some experiments, mice were treated with control or Mogat1 or control ASOs in the presence or absence of IFNAR-1 neutralizing antibody.
Results
Genetic deletion of hepatic Mogat1 , either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced weight gain, insulin resistance, and glucose intolerance on a HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression hepatocytes increased liver MGAT activity and TAG content in low-fat fed mice, but did not cause insulin resistance. Interestingly, Mogat1 ASO treatment improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off target effect. Inhibition of IFNAR-1 did not block the effect of Mogat1 ASO on glucose homeostasis.
Conclusion
These results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling.
Abstract Figure
Highlights
Mogat1 liver-specific KO or KD does not improve metabolism in HFD fed mice.
Whole-body Mogat1-deletion impairs insulin tolerance in HFD fed mice.
Mogat1 ASOs improves whole body metabolism independently of gene knockdown.
Blockade of the INFR response does not prevent off-target effects of Mogat1 ASOs.
... While MGAT1 expression is absent in the small intestine (Cases et al., 1998(Cases et al., , 2001Yen et al., 2002), MGAT2 expression is abundant in mouse gut (Yen et al., 2002) and its activity correlates with the rate of MG absorption (Yen et al., 2015). However, the deletion of MGAT2 did not result in a change in normal quantities of fat absorbed from the small intestine aside from an increased energy expenditure noted in knockout mice (Yen et al., 2009;Nelson et al., 2011). ...
... While MGAT1 expression is absent in the small intestine (Cases et al., 1998(Cases et al., , 2001Yen et al., 2002), MGAT2 expression is abundant in mouse gut (Yen et al., 2002) and its activity correlates with the rate of MG absorption (Yen et al., 2015). However, the deletion of MGAT2 did not result in a change in normal quantities of fat absorbed from the small intestine aside from an increased energy expenditure noted in knockout mice (Yen et al., 2009;Nelson et al., 2011). ...
During the last two decades, a large body of information on the events responsible for intestinal fat digestion and absorption has been accumulated. In particular, many groups have extensively focused on the absorptive phase in order to highlight the critical “players” and the main mechanisms orchestrating the assembly and secretion of chylomicrons (CM) as essential vehicles of alimentary lipids. The major aim of this article is to review understanding derived from basic science and clinical conditions associated with impaired packaging and export of CM. We have particularly insisted on inborn metabolic pathways in humans as well as on genetically modified animal models (recapitulating pathological features). The ultimate goal of this approach is that “experiments of nature” and in vivo model strategy collectively allow gaining novel mechanistic insight and filling the gap between the underlying genetic defect and the apparent clinical phenotype. Thus, uncovering the cause of disease contributes not only to understanding normal physiologic pathway, but also to capturing disorder onset, progression, treatment and prognosis.
... However, a challenge with sn-2 structures is that they are not very stable and easily undergo isomerization to form sn-1-(3)-MAG structures. Interestingly, OM3 was shown to be an effective substrate to MGAT enzyme and could be used as a pre-digested fat [37,38] with potentially improved bioavailability compared to ethyl ester or TAG. This lipase-independent MAG carrier may offer a clinical advantage to patients struggling with fat malabsorption/maldigestion problems, for instance, patients with cystic fibrosis [21,[37][38][39]. ...
... Interestingly, OM3 was shown to be an effective substrate to MGAT enzyme and could be used as a pre-digested fat [37,38] with potentially improved bioavailability compared to ethyl ester or TAG. This lipase-independent MAG carrier may offer a clinical advantage to patients struggling with fat malabsorption/maldigestion problems, for instance, patients with cystic fibrosis [21,[37][38][39]. ...
Numerous benefits are attributed to omega-3 fatty acids (OM3) especially in cardiovascular health. However, bioavailability and clinical efficacy depend on numerous factors, including OM3 form, food matrix effects (especially the lipid content of the diet), and metabolic capacity. Here, we show in humans that a “pre-digested” OM3-sn-1(3)-monoacylglycerol lipid structure (OM3-MAG) has a significantly greater absorption at high therapeutic doses (2.9 g/day) than the most commonly OM3-ethyl ester (3.1 g/day) form (used for the treatment of hypertriglyceridemia), and a comparable profile to other pre-digested OM3 free fatty acids (OM3-FFA) structure (3.2 g/day). Nutritional supplement doses of MAG resulted in similar increases in OM3 blood level, compared to OM3 triacylglycerols (OM3-TAG) supplements in obese subjects (1.2 g/day) under low fat diet, and in children with cystic fibrosis (1.0 g/day). These results suggest that both forms of pre-digested OM3-MAG and OM3-FFA are effectively absorbed and re-incorporated effectively into triacylglycerols inside the enterocytes, before being exported into the chylomicrons lipid transport system. The pre-digested OM3-MAG might provide a more effective therapy in severe cardiovascular conditions where high doses of OM3 are required and a low-fat diet is indicated, which limited digestive lipase activity.
... Alternatively, monoacylglycerol (MAG), which is composed of one fatty acid and a glycerol backbone (unphosphorylated), can be directly converted into DAG through acylation by monoacylglycerol acyltransferases (MGATs, gene symbol Mogats) (1,2). The MGAT pathway is highly active in intestine, where it is involved in dietary fat absorption (3)(4)(5). ...
... Subsequent publications suggest MGAT activity spares essential fatty acids from oxidation by re-esterifying these lipids in the context of lipolytic stimuli (9, 33). In the original studies, MGAT1 was shown to prefer unsaturated long-chain acyl-CoAs as a substrate (5). Whether one of these essential fatty acids or complex lipids is serving as an endogenous ligand for PPARα or another complex lipid downstream of MGAT activity remains to be determined. ...
During prolonged fasting, the liver plays a central role in maintaining systemic energy homeostasis by producing glucose and ketones in processes fueled by oxidation of fatty acids liberated from adipose tissue. In mice, this is accompanied by transient hepatic accumulation of glycerolipids. We found that the hepatic expression of monoacylglycerol acyltransferase 1 (Mogat1), an enzyme with monoacylglycerol acyltransferase (MGAT) activity that produces diacyl-glycerol from monoacylglycerol, was significantly increased in the liver of fasted mice compared with mice given ad libitum access to food. Basal and fasting-induced expression of Mogat1 was markedly diminished in the liver of mice lacking the transcription factor PPARα. Suppressing Mogat1 expression in liver and adipose tissue with antisense oligonucleotides (ASOs) reduced hepatic MGAT activity and triglyceride content compared with fasted controls. Surprisingly, the expression of many other PPARα target genes and PPARα activity was also decreased in mice given Mogat1 ASOs. When mice treated with control or Mogat1 ASOs were gavaged with the PPARα ligand, WY-14643, and then fasted for 18 h, WY-14643 administration reversed the effects of Mogat1 ASOs on PPARα target gene expression and liver triglyceride content. In conclusion, Mogat1 is a fasting-induced PPARα target gene that may feed forward to regulate liver PPARα activity during food deprivation.
... Three related MGAT-encoding genes have been identified in humans; namely MOGAT1, MOGAT2, and MOGAT3, which encode MGAT1, MGAT2 and MGAT3. In mice, Mogat1 and Mogat2 encode MGAT1 and MGAT2, but the mouse Mogat3 is a pseudogene and not analogous to human MOGAT3 (7)(8)(9). Data from a series of studies have shown that MGAT2 is important for enterocyte absorption of ingested fat (7,(10)(11)(12). MGAT1 is not well-expressed in intestine, but MGAT1-mediated MGAT activity may be important in the pathogenesis of obesity-related hepatic insulin resistance (13)(14)(15). ...
... Besides intestine and liver, the metabolic effects of MGAT enzymes have been little studied. For example, although MGAT expression and activity have been detected in adipose tissue (8,17), its contribution to adipocyte lipid metabolism is unclear. ...
Adipocyte triglyceride storage provides a reservoir of energy that allows the organism to survive times of nutrient scarcity, but excessive adiposity has emerged as a health problem in many areas of the world. Monoacylglycerol acyltransferase (MGAT) acylates monoacylglycerol to produce diacylglycerol; the penultimate step in triglyceride synthesis. However, little is known about MGAT activity in adipocytes, which are believed to rely primarily on another pathway for triglyceride synthesis. We show that expression of the gene that encodes MGAT1 is robustly induced during adipocyte differentiation and that its expression is suppressed in fat of genetically-obese mice and metabolically-abnormal obese human subjects. Interestingly, MGAT1 expression is also reduced in physiologic contexts where lipolysis is high. Moreover, knockdown or knockout of MGAT1 in adipocytes leads to higher rates of basal adipocyte lipolysis. Collectively, these data suggest that MGAT1 activity may play a role in regulating basal adipocyte fatty acid retention.
... There are three isoforms of monoacylglycerol acyltransferases (Mogat 1-3). Mogat1 is primarily expressed in the intestines (enterocytes of the small intestines) followed by the stomach and kidney, 15 as well as in conditions of liver pathology, such as hepatic steatosis. 9 The findings described here are part of a larger study where we demonstrate the regeneration of AT in Agpat2 À/À mice, 16 overexpression of human AGPAT2 (hAGPAT2) specifically in AT, and the effect of AGPAT2 on TAG synthesis in AT. 17 To investigate this, we constructed a transgenic murine model expressing hAGPAT2 specifically in AT, driven by the adiponectin promoter, and regulated by doxycycline (dox) 16 (Tg-AT-hA2 ;mA2 À/À ) (Figures S1A and S1B). ...
Both humans and mice with congenital generalized lipodystrophy due to AGPAT2 deficiency develop diabetes mellitus, insulin resistance, and hepatic steatosis, which have been attributed to the near total loss of adipose tissue (AT). Here, we show that regulated AT regeneration in doxycycline (dox)-fed Tg−AT-hAGPAT2;mAgpat2−/− mice partially ameliorates hepatic steatosis at 12 weeks of age and causes reduced expression of genes involved in hepatic de novo lipogenesis despite partial (∼30–50%) AT regeneration compared to that in wild-type mice. Compared to chow-fed Tg−AT-hAGPAT2;mAgpat2−/− mice, those fed dox diet had markedly reduced serum insulin levels, suggesting an improvement in insulin resistance. Interestingly, the fasting plasma glucose levels in dox-fed Tg−AT-hAGPAT2;mAgpat2−/− mice were no different than those in chow-fed wild-type mice. Indirect calorimetry revealed normalization in the energy balance of dox-fed Tg−AT-hAGPAT2;mAgpat2−/− mice compared to that in chow-fed mice. This study’s findings suggest that partial AT regeneration in lipodystrophic mice can ameliorate metabolic derangements.
... Another pathway involved in triglyceride synthesis is the glycerol 3-phosphate pathway [8]. Among the three MOGAT enzymes, only MOGAT2 (also known as MGAT2) is highly expressed in the intestine of both mice and humans [9][10][11][12]. ...
Background: Dietary fat absorption involves the re-esterification of digested triacylglycerol in the enterocytes, it is a biological process catalyzed by monoacylglycerol O-acyltransferase 2 (MOGAT2, aka MGAT2), which is highly expressed in the small intestine. A previous study showed that the loss of the Mogat2 gene can prevent high-fat diet-induced obesity in mice. Obesity is associated with an increased risk of several types of cancer including a postmenopausal mammary tumor. Methods: We collected 147 patients with triple-negative breast adenocarcinoma to explore the relationship between MOGAT2 expression and overall patient survival. The TCGA data were also retrieved for analyzing the prognostic values of MOGAT2 mRNA level as well as the relationships between MOGAT2 and DGAT1/2 mRNA levels. We also used a Mogat2-deficient mouse mammary tumor model by crossing Mogat2-deficient mice with MMTV-PyMT mice to examine the effect of MOGAT2 on mammary tumor development. Results: In human triple-negative breast adenocarcinoma, elevated expression of MOGAT2 correlated with a poorer patient prognosis. Obesity could be induced by a relatively high-fat diet (37% of calories from fat) in the mice with or without Mogat2 knockout. Mammary tumor development was deteriorated by a relatively high-fat diet regardless of Mogat2 deficiency. As a compensation mechanism, upregulation of diacylglycerol O-acyltransferases 1 and 2 (Dgat1 and Dgat2) in the Mogat2 deficient mice was found. Consistently, in human normal tissues adjacent to breast cancer, an inverse correlation between MOGAT2 mRNA level and DGAT1/2 mRNA levels was also found. Conclusions: Elevated expression of MOGAT2 in triple-negative breast adenocarcinoma predicts poorer patient overall survival. With the compensation of Dgat1 and Dgat2, Mogat2 deficiency alone cannot prevent fat diet-induced obesity, nor prevent mammary tumor development in a mouse model.
... However, the increase in liver TAG induced by an HFD in control animals was prevented in 9A-MGS Alb mice ( Figure 5A). The lower hepatic TAG content in 9A-MGS Alb mice fed an HFD was associated with a decrease in the expression of monoacylglycerol acyltransferase 1 (MGAT1) ( Figure 5B), a microsomal enzyme that catalyzes the synthesis of diacylglycerol and TAG [17]. MGAT1 co-localizes to lipid droplets under conditions of enriching fatty acids, thereby contributing to TAG synthesis [18]. ...
Many lines of evidence demonstrate a correlation between liver glycogen content and food intake. We previously demonstrated that mice overexpressing protein targeting to glycogen (PTG) specifically in the liver—which have increased glycogen content in this organ—are protected from high-fat diet (HFD)-induced obesity by reduced food intake. However, the use of PTG to increase liver glycogen implies certain limitations. PTG stimulates glycogen synthesis but also inhibits the enzyme responsible for glycogen degradation. Furthermore, as PTG is a regulatory subunit of protein phosphatase 1 (PP1), which regulates many cellular functions, its overexpression could have side effects beyond the regulation of glycogen metabolism. Therefore, it is necessary to determine whether the direct activation of glycogen synthesis, without affecting its degradation or other cellular functions, has the same effects. To this end, we generated mice overexpressing a non-inactivatable form of glycogen synthase (GS) specifically in the liver (9A-MGSAlb mice). Control and 9a-MGSAlb mice were fed a standard diet (SD) or HFD for 16 weeks. Glucose tolerance and feeding behavior were analyzed. 9A-MGSAlb mice showed an increase in hepatic glycogen in fed and fasting conditions. When fed an HFD, these animals preserved their hepatic energy state, had a reduced food intake, and presented a lower body weight and fat mass than control animals, without changes in energy expenditure. Furthermore, 9A-MGSAlb animals showed improved glucose tolerance when fed an SD or HFD. Moreover, liver triacylglycerol levels that were increased after HFD feeding were lower in these mice. These results confirm that increased liver glycogen stores contribute to decreased appetite and improve glucose tolerance in mice fed an HFD. On the basis of our findings, strategies to preserve hepatic glycogen stores emerge as potential treatments for obesity and hyperglycemia.
... Figure 5b)(Kumari et al., 2012;Prasad et al., 2011;Shulga et al., 2011;C. L. E. Yen et al., 2002), phosphatidylcholine and phosphatidylethanolamine metabolism (Figure 5c)(Cao et al., 2008;Golczak et al., 2012;Horibata & Hirabayashi, 2007;Payton et al., 2004;Schaloske & Dennis, 2006;Tsuboi et al., 2015), and the metabolism of membrane sphingolipids (Figure 5d) (T. J.Kim et al., 2006;Lahiri et al., 2007;Tafesse et al., 2007;Takahashi ...
This study aimed to identify transcriptome differences between distinct or transitional stage spherical, ovoid, and tubular porcine blastocysts throughout the initiation of elongation. We performed a global transcriptome analysis of differential gene expression using RNA-Seq with high temporal resolution between spherical, ovoid, and tubular stage blastocysts at specific sequential stages of development from litters containing conceptus populations of distinct or transitional blastocysts. After RNA-Seq analysis, significant differentially expressed genes (DEGs) and pathways were identified between distinct morphologies or sequential development stages. Overall, 1898 significant DEGs were identified between distinct spherical and ovoid morphologies, with 311 total DEGs between developmental stages throughout this first morphological transition, while 15 were identified between distinct ovoid and tubular, with eight total throughout these second morphological transition developmental stages. The high quantity of DEGs and pathways between conceptus stages throughout the spherical to ovoid transition suggests the importance of gene regulation during this first morphological transition for initiating elongation. Further, extensive DEG coverage of known elongation signaling pathways was illustrated from spherical to ovoid, and regulation of lipid signaling and membrane/ECM remodeling across these early conceptus stages were implicated as essential to this process, providing novel insights into potential mechanisms governing this rapid morphological change.
... The optimized YSK05-LNPs were also applied to the in vivo validation of candidate genes as therapeutic targets. 55) A comparative transcriptome analysis in liver between diabetic and normal mice identified an elevated expression of monoacylglycerol O-acyltransferase 1 (Mogat-1), an enzyme that is involved in triglyceride synthesis and its storage. [56][57][58] Long term Mogat-1 gene silencing by the repeated administration of siMogat-1-loaded YSK05-LNPs in diabetic mice resulted in preventive effects of type 2 diabetes, including reduced blood glucose levels, triglycerides and cholesterol, and increased levels of adiponectin. ...
Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.
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... Another pathway involved in triglyceride synthesis is the glycerol 3-phosphate pathway (8). Among the three MOGAT enzymes, only MOGAT2 (also known as MGAT2) is highly expressed in the intestine of both mice and humans (9)(10)(11)(12). ...
Background: Dietary fat absorption involves the re-esterification of digested triacylglycerol in the enterocytes, it is a biological process catalyzed by monoacylglycerol O-acyltransferase 2 (MOGAT2, aka MGAT2), which is highly expressed in the small intestine. A previous study showed that the loss of the Mogat2 gene can prevent high-fat diet-induced obesity in mice. Obesity is associated with an increased risk of several types of cancer including postmenopausal breast cancer.
Methods: We collected 147 patients with triple negative breast adenocarcinoma to explore the relationship between the expression of MOGAT2 and patient overall survival. And we generated a Mogat2-deficient mouse mammary tumor model by crossing Mogat2-deficient mice with MMTV-PyMT mice to examine the effect of losing MOGAT2 in vivo.
Results: Our founding suggest that obesity was induced by a relatively high-fat diet (37% of calories from fat) in the mice with or without Mogat2 knockout. Mammary tumor development was deteriorated by a relatively high-fat diet regardless of Mogat2 deficiency. As a compensation mechanism, upregulation of diacylglycerol O-acyltransferases 1 and 2 (Dgat1 and Dgat2) in the Mogat2 deficient mice was found.
Conclusions: Elevated expression of MOGAT2 in triple negative breast adenocarcinoma predicts poorer patient overall survival. With the compensation of Dgat1 and Dgat2, Mogat2 deficiency alone cannot prevent fat diet-induced obesity, nor prevent mammary tumor development in a mouse model.
... In addition to PKC, DG regulates a wide variety of signal transduction proteins, such as b2-chimaerin, protein kinase D, Ras guanyl nucleotide-releasing protein and Unc-13 (3)(4)(5)(6). In eukaryotes, DG is generated from two major lipids: the glycerolipids, such as monoacylglycerol and triacylglycerol (7)(8)(9) via acyltransferase and lipase, respectively, and glycerophospholipids, such as phosphatidic acid (PA), phosphatidylinositol 4,5bisphosphate (PI(4,5)P2), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) via PA phosphatase (PAP)/lipid phosphate phosphatase (LPP) or phospholipase C (PLC) (10)(11)(12)(13). Although type II PAP/lipid phosphate phosphatase (PAP2/LPP), lipin (type I PAP) and PI(4,5)P2specific phospholipase C (PLC), as DG-generating enzymes, have been cloned and extensively studied in mammals (10,14,15), the molecular entities (the genes and proteins) of PC-and PE-specific PLCs have not been identified until now (12,16). ...
Diacylglycerol (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid-containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor-dependent cell proliferation, type 2 diabetes and obsessive-compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity via hydrolysis of PE, phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylcholine (PC) in the absence of ceramide. DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PI(4,5)bisphosphate (PI(4,5)P2). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PA molecular species, but not arachidonic acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PI(4,5)P2 cycle.
... Considering that the genus Nostoc has been reported to accumulate lipid bodies (Peramuna and Summers 2014), RS25730 may mainly work as DGAT. Mammalian acyl-CoA:monoacylglycerol acyltransferase enzymes have significant sequence similarity with DGAT2 enzymes but preferentially acylate monoacylglycerols (Yen et al. 2002). Aizouq et al. (2020) pointed out that Slr2103 is homologous to the C-terminal domain of PES1 and PES2 in Arabidopsis shown in orange in Fig. 7. ...
Although cyanobacteria do not possess wax ester synthase/acyl-CoA: diacylglycerol acyltransferase (WS/DGAT), the bacterial enzyme for triacylglycerol (TAG) production, there have been several studies reporting accumulation of TAG-like compounds in cyanobacteria. In this study, we aimed to evaluate TAG productivity of the ΔrecJ::atfA strain of Synechocystis sp. PCC 6803 generated by inserting atfA encoding WS/DGAT from Acinetobacter baylyi ADP1 into recJ (sll1354), together with the wild type (WT) and the gene-disrupted strain of slr2103 having homology with eukaryotic DGAT2 gene family (Δ2103). Thin layer chromatography (TLC) of neutral lipids or isolation of the neutral lipids-enriched fraction followed by gas chromatography or liquid chromatography-tandem mass spectrometry was employed for analyses. The ΔrecJ::atfA strain accumulated 0.508 nmol mL-1OD730-1 of TAG after a week of incubation at 100 μmol photons m-2s-1. The saturated fatty acids C16:0 and C18:0 accounted for about 50% and 20% of the TAG fatty acids, respectively, suggesting that de novo-synthesized fatty acids were preferentially incorporated into TAG molecules. When the neutral lipid profile of the lipid extracts was examined by TLC, a spot located slightly lower position compared with TAG standard was detected in WT but not in the Δ2103 strain. TAG accumulation level of both strains was only 0.01∼0.03 nmol mL-1 OD730-1, but the fatty acid composition was substantially different from that of the background. These results suggest trace amounts of TAG can be produced in Synechocystis cells by other enzymes than Slr2103 and major constituents of the TAG-like spot are unknown lipid species produced by Slr2103.
... Mgat1 is reportedly expressed in the stomach, kidney, and adipose tissue, and at low levels in the liver. On the other hand, Mgat2 is predominantly expressed in the small intestine (Cao et al., 2003(Cao et al., , 2004Yen and Farese, 2003;Yen et al., 2002). In our studies, Mgat1 mRNA levels were very low in the livers of wild-type mice (Ct value > 30); however, the deletion of Agpat2 was associated with a robust 25-to 48-fold induction of Mgat1 mRNA (Table 2) and $5to 7-fold increase in MGAT1 hepatic protein ( Figure 2D), suggesting that enhanced TG synthesis can occur via this alternate MAG pathway in the livers of Agpat2 À/À mice, such as we previously proposed (Agarwal and Garg, 2003). ...
Mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) cause congenital generalized lipodystrophy. To understand the molecular mechanisms underlying the metabolic complications associated with AGPAT2 deficiency, Agpat2 null mice were generated. Agpat2(-/-) mice develop severe lipodystrophy affecting both white and brown adipose tissue, extreme insulin resistance, diabetes, and hepatic steatosis. The expression of lipogenic genes and rates of de novo fatty acid biosynthesis were increased approximately 4-fold in Agpat2(-/-) mouse livers. The mRNA and protein levels of monoacylglycerol acyltransferase isoform 1 were markedly increased in the livers of Agpat2(-/-) mice, suggesting that the alternative monoacylglycerol pathway for triglyceride biosynthesis is activated in the absence of AGPAT2. Feeding a fat-free diet reduced liver triglycerides by approximately 50% in Agpat2(-/-) mice. These observations suggest that both dietary fat and hepatic triglyceride biosynthesis via a monoacylglycerol pathway may contribute to hepatic steatosis in Agpat2(-/-) mice.
... Although there is no evidence of association between MGAT1 and lipids as reported in prior studies of non-Pacific Islanders, variation near MGAT1 has been associated with BMI, serum fatty acid levels and composition, and glucose response in Europeans [36][37][38] . The encoded MGAT enzyme plays a major role in the absorption of dietary fat in the intestine 39 . Due to the lower frequency of the HDL-associated risk variant observed in Samoans compared to 1000 ...
Background: The current understanding of the genetic architecture of lipids have largely come from genome-wide association studies. To date, few studies have examined the genetic architecture of lipids in Polynesians, and none have in Samoans, whose unique population history, including many population bottlenecks, may provide insight into the biological foundations of variation in lipid levels.
Methods and Results: Here we performed a genome-wide association study of four fasting serum lipid levels: total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglycerides (TG) in a sample of 2,849 Samoans, with validation genotyping for associations in a replication cohort comprising 1,798 Samoans and American Samoans. We identified multiple genome-wide significant associations (P < 5 x 10-8) previously seen in other populations - APOA1 with TG, CETP with HDL, and APOE with TC and LDL - and several suggestive associations (P < 1 x 10-5), including an association of variants downstream of MGAT1 and RAB21 with HDL. For variants near APOE and APOA1, we observed different association signals than what has been previously reported in non-Polynesian populations.
Conclusions: The association with several known lipid loci combined with the newly-identified associations with variants near MGAT1 and RAB21 suggest that while some of the genetic architecture of lipids is shared between Samoans and other populations, part of the genetic etiology may be Polynesian-specific.
... And, HFD led to the upward adjustment of fatty acid uptake and lipid storage via the gene expression of PPARg and CD36, DBDPE enhanced this pathway by increasing the expression of PPARg in the LFD group. After exposure to DBDPE, PPARa, Cpt1a and Mttp had down-regulation trend, which meant DBDPE may inhibit the b-oxidation of fatty acid and triglyceride transport ( Zhang et al., 2017), and the up-regulation of Mogat1, an enzyme that regulates triglyceride synthesis ( Yen et al., 2002), might also be the cause of obesity in mice. Simultaneously, the changes of G6pase and Gck also caused disorders of glucose metabolism, which in turn aggravated obesity in mice, while the joint exposure might lead to different effects compared with exposure alone. ...
... We next attempted to understand the molecular mechanisms contributing to hepatic steatosis and insulin resistance in SL mice. We found major alterations in expression of genes of the MOGAT pathway, which might play a key role in mediating both hepatic steatosis and insulin resistance in our mouse model (Fig. 6B) (38). Combined data from SL, control, and LL mice showed that Mogat1 expression correlated positively with hepatic TAG concentration. ...
Postnatal overfeeding increases the risk of chronic diseases later in life, including obesity, insulin resistance, hepatic steatosis, and type 2 diabetes. Epigenetic mechanisms might underlie the long-lasting effects associated with early nutrition. Here we aimed to explore the molecular pathways involved in early development of insulin resistance and hepatic steatosis, and we examined the potential contribution of DNA methylation and histone modifications to long-term programming of metabolic disease. We used a well-characterized mouse model of neonatal overfeeding and early adiposity by litter size reduction. Neonatal overfeeding led to hepatic insulin resistance very early in life that persisted throughout adulthood despite normalizing food intake. Up-regulation of monoacylglycerol O-acyltransferase ( Mogat) 1 conceivably mediates hepatic steatosis and insulin resistance through increasing intracellular diacylglycerol content. Early and sustained deregulation of Mogat1 was associated with a combination of histone modifications that might favor Mogat1 expression. In sum, postnatal overfeeding causes extremely rapid derangements of hepatic insulin sensitivity that remain relatively stable until adulthood. Epigenetic mechanisms, particularly histone modifications, could contribute to such long-lasting effects. Our data suggest that targeting hepatic monoacylglycerol acyltransferase activity during early life might provide a novel strategy to improve hepatic insulin sensitivity and prevent late-onset insulin resistance and fatty liver disease.-Ramon-Krauel, M., Pentinat, T., Bloks, V. W., Cebrià, J., Ribo, S., Pérez-Wienese, R., Vilà, M., Palacios-Marin, I., Fernández-Pérez, A., Vallejo, M., Téllez, N., Rodríguez, M. À., Yanes, O., Lerin, C., Díaz, R., Plosch, T., Tietge, U. J. F., Jimenez-Chillaron, J. C. Epigenetic programming at the Mogat1 locus may link neonatal overnutrition with long-term hepatic steatosis and insulin resistance.
Non‐alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease. If not treated, it can lead to liver damage, cirrhosis and even liver cancer. However, advances in treatment have remained relatively slow, and there is thus an urgent need to develop appropriate treatments. Hedan tablet (HDP) is used to treat metabolic syndrome. However, scientific understanding of the therapeutic effect of HDP on NASH remains limited. We used HDP to treat a methionine/choline‐deficient diet‐induced model of NASH in rats to elucidate the therapeutic effects of HDP on liver injury. In addition, we used untargeted metabolomics to investigate the effects of HDP on metabolites in liver of NASH rats, and further validated its effects on inflammation and lipid metabolism following screening for potential target pathways. HDP had considerable therapeutic, anti‐oxidant, and anti‐inflammatory effects on NASH. HDP could also alter the hepatic metabolites changed by NASH. Moreover, HDP considerable moderated NF‐κB and lipid metabolism‐related pathways. The present study found that HDP had remarkable therapeutic effects in NASH rats. The therapeutic efficacy of HDP in NASH mainly associated with regulation of NF‐κB and lipid metabolism‐related pathways via arachidonic acid metabolism, glycine‐serine‐threonine metabolism, as well as steroid hormone biosynthesis.
Background
The identification of cell type-specific genes and their modification under different conditions is central to our understanding of human health and disease. The stomach, a hollow organ in the upper gastrointestinal tract, provides an acidic environment that contributes to microbial defence and facilitates the activity of secreted digestive enzymes to process food and nutrients into chyme. In contrast to other sections of the gastrointestinal tract, detailed descriptions of cell type gene enrichment profiles in the stomach are absent from the major single-cell sequencing-based atlases.
Results
Here, we use an integrative correlation analysis method to predict human stomach cell type transcriptome signatures using unfractionated stomach RNAseq data from 359 individuals. We profile parietal, chief, gastric mucous, gastric enteroendocrine, mitotic, endothelial, fibroblast, macrophage, neutrophil, T-cell, and plasma cells, identifying over 1600 cell type-enriched genes.
Conclusions
We uncover the cell type expression profile of several non-coding genes strongly associated with the progression of gastric cancer and, using a sex-based subset analysis, uncover a panel of male-only chief cell-enriched genes. This study provides a roadmap to further understand human stomach biology.
Plasmalogen is a major phospholipid of mammalian cell membranes. Recently it is becoming evident that the sn-1 vinyl-ether linkage in plasmalogen, contrasting to the ester linkage in the counterpart diacyl glycerophospholipid, yields differential molecular characteristics for these lipids especially related to hydrocarbon-chain order, so as to concertedly regulate biological membrane processes. A role played by NMR in gaining information in this respect, ranging from molecular to tissue levels, draws particular attention. We note here that a broad range of enzymes in de novo synthesis pathway of plasmalogen commonly constitute that of diacyl glycerophospholipid. This fact forms the basis for systematic crosstalk that not only controls a quantitative balance between these lipids, but also senses a defect causing loss of lipid in either pathway for compensation by increase of the counterpart lipid. However, this inherent counterbalancing mechanism paradoxically amplifies imbalance in differential effects of these lipids in a diseased state on membrane processes. While sharing of enzymes has been recognized, it is now possible to overview the crosstalk with growing information for specific enzymes involved. The overview provides a fundamental clue to consider cell and tissue type-dependent schemes in regulating membrane processes by plasmalogen and diacyl glycerophospholipid in health and disease.
Objective:
Hepatic triacylglycerol accumulation and insulin resistance are key features of NAFLD. However, NAFLD development and progression are rather triggered by the aberrant generation of lipid metabolites and signaling molecules including diacylglycerol (DAG) and lysophosphatidylcholine (lysoPC). Recent studies showed decreased expression of carboxylesterase 2 (CES2) in the liver of NASH patients and hepatic DAG accumulation was linked to low CES2 activity in obese individuals. The mouse genome encodes several Ces2 genes with Ces2a showing highest expression in the liver. Herein we investigated the role of mouse Ces2a and human CES2 in lipid metabolism in vivo and in vitro.
Methods:
Lipid metabolism and insulin signaling were investigated in mice lacking Ces2a and in a human liver cell line upon pharmacological CES2 inhibition. Lipid hydrolytic activities were determined in vivo and from recombinant proteins.
Results:
Ces2a deficient mice (Ces2a-ko) are obese and feeding a high-fat diet (HFD) provokes severe hepatic steatosis and insulin resistance together with elevated inflammatory and fibrotic gene expression. Lipidomic analysis revealed a marked rise in DAG and lysoPC levels in the liver of Ces2a-ko mice fed HFD. Hepatic lipid accumulation in Ces2a deficiency is linked to lower DAG and lysoPC hydrolytic activities in liver microsomal preparations. Moreover, Ces2a deficiency significantly increases hepatic expression and activity of MGAT1, a PPAR gamma target gene, suggesting aberrant lipid signaling upon Ces2a deficiency. Mechanistically, we found that recombinant Ces2a and CES2 show significant hydrolytic activity towards lysoPC (and DAG) and pharmacological inhibition of CES2 in human HepG2 cells largely phenocopies the lipid metabolic changes present in Ces2a-ko mice including reduced lysoPC and DAG hydrolysis, DAG accumulation and impaired insulin signaling.
Conclusions:
Ces2a and CES2 are critical players in hepatic lipid signaling likely via the hydrolysis of DAG and lysoPC at the ER.
Tetraspanins, a superfamily of membrane proteins, mediate diverse biological processes through tetraspanin-enriched microdomains in the plasma membrane. However, how their cell-surface presentation is controlled remains unclear. To identify the regulators of tetraspanin trafficking, we conduct sequential genome-wide loss-of-function CRISPR-Cas9 screens based on cell-surface expression of a tetraspanin member, TSPAN8. Several genes potentially involved in endoplasmic reticulum (ER) targeting, different biological processes in the Golgi apparatus, and protein trafficking are identified and functionally validated. Importantly, we find that biantennary N-glycans generated by MGAT1/2, but not more complex glycan structures, are important for cell-surface tetraspanin expression. Moreover, we unravel that SPPL3, a Golgi intramembrane-cleaving protease reported previously to act as a sheddase of multiple glycan-modifying enzymes, controls cell-surface tetraspanin expression through a mechanism associated with lacto-series glycolipid biosynthesis. Our study provides critical insights into the molecular regulation of cell-surface presentation of tetraspanins with implications for strategies to manipulate their functions, including cancer cell invasion.
L’intestin joue un rôle clé dans le contrôle de l’homéostasie énergétique. Les entérocytes sont des cellules polarisées qui permettent les échanges entre la lumière intestinale (membrane apicale) et le compartiment lymphatique et sanguin (membrane baso-latérale). Dans cette thèse, nous nous sommes particulièrement intéressés au contrôle par les entérocytes de deux processus liés au métabolisme des lipides et du cholestérol : l’excrétion trans-intestinale de cholestérol (TICE) et l’absorption des lipides alimentaires.Très récemment, il a été montré que l’intestin contribue à 20-30% de l’excrétion fécale du cholestérol chez la souris. Ce mécanisme, appelé TICE, implique le passage direct du cholestérol provenant de la circulation sanguine à travers les entérocytes vers les fèces. De par son caractère modulable par des substances pharmacologiques comme l’ézétimibe et les statines, le TICE représente une cible thérapeutique potentielle pour corriger les dyslipidémies athérogènes du diabétique. Cependant, les mécanismes moléculaires gouvernant le transport rétrograde du cholestérol (du pôle baso-latéral au pôle apical) dans l’entérocyte lors du TICE, sont complètement inconnus. Dans une première étude, nous avons mis en évidence la lignée entérocytaire humaine Caco-2/TC7 comme un modèle d’étude des processus trans-entérocytaires liés au TICE. Nous avons d’abord montré que suite à l’incubation avec du plasma humain dans le compartiment baso-latéral et des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 miment des caractéristiques du TICE in vivo. De plus, grâce à ce modèle in vitro, nous avons pu identifier les microtubules comme acteurs nécessaires au transport rétrograde du cholestérol dans l’entérocyte. Dans une seconde étude, nous nous sommes intéressés au contrôle par le récepteur nucléaire Rev-erbα de la production des chylomicrons (CM) par les entérocytes. En effet, bien qu’essentiellement vue comme la conséquence d’une clairance retardée, des données émergentes présentent la surproduction de CM par l’intestin comme un contributeur majeur de la dyslipidémie chez l’insulino-résistant. Il existe une balance, au sein de l’entérocyte, entre l’utilisation des lipides absorbés pour un stockage transitoire sous forme de gouttelettes lipidiques (GL) cytosoliques ou pour l’assemblage de lipoprotéines riches en triglycérides (LRT). Le récepteur nucléaire Rev-erbα est un répresseur transcriptionnel impliqué dans le métabolisme énergétique et le rythme circadien. Rev-erbα contrôle particulièrement le métabolisme lipidique au niveau du foie et le catabolisme des LRT. Pour cette seconde étude, une lignée Caco-2/TC7 invalidée pour Rev-erbα (sh Rev-erbα) a donc été développée par infection lentivirale et différenciée sur insert. Les résultats indiquent que suite à l’incubation avec des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 sh Rev-erbα sécrètent plus de LRT dans le milieu baso-latéral et stockent moins de lipides sous la forme de GL cytosoliques. De plus, la lignée Caco-2/TC7 sh Rev-erbα présente une activité lipophagique plus importante et l’inhibition de l’autophagie par la bafilomycine dans cette lignée restaure la sécrétion baso-latérale de LRT et le stockage intracellulaire de GL aux mêmes niveaux que ceux de la lignée sh control. Cette seconde étude montre donc que l’invalidation de Rev-erbα dans l’entérocyte entraîne une augmentation de la mobilisation des lipides des GL via le processus de la lipophagie résultant en une augmentation de la sécrétion de LRT. Notre hypothèse est que Rev-erbα joue un rôle clé dans le contrôle de la balance GL/LRT et donc de la triglycéridémie post-prandiale.Les deux études présentées dans cette thèse permettent une meilleure compréhension des mécanismes liés au contrôle du métabolisme lipidique par l’intestin et mettent ainsi en avant l’intestin comme une cible thérapeutique potentielle pour corriger les dyslipidémies du diabétique.
Objective:
Monoacylglycerol O-acyltransferase 1 (Mogat1), a lipogenic enzyme that converts monoacylglycerol to diacylglycerol, is highly expressed in adipocytes and may regulate lipolysis by re-esterifying fatty acids released during times when lipolytic rates are low. However, the role of Mogat1 in regulating adipocyte fat storage during differentiation and diet-induced obesity is relatively understudied.
Methods:
Here, adipocyte-specific Mogat1 knockout mice were generated and subjected to a high-fat diet to determine the effects of Mogat1 deficiency on diet-induced obesity. Mogat1 floxed mice were also used to develop preadipocyte cell lines wherein Mogat1 could be conditionally knocked out to study adipocyte differentiation in vitro.
Results:
In preadipocytes, it was found that Mogat1 knockout at the onset of preadipocyte differentiation prevented the accumulation of glycerolipids and reduced the differentiation capacity of preadipocytes. However, the loss of adipocyte Mogat1 did not affect weight gain or fat mass induced by a high-fat diet in mice. Furthermore, loss of Mogat1 in adipocytes did not affect plasma lipid or glucose concentrations or insulin tolerance.
Conclusions:
These data suggest Mogat1 may play a role in adipocyte differentiation in vitro but not adipose tissue expansion in response to nutrient overload in mice.
The members of the monoacylglycerol acyltransferase (MOGAT) family are essential candidate genes that influence economic traits associated with triglyceride synthesis, dietary fat absorption, and storage in livestock. In addition, the MOGAT gene family may also play an essential function in human polygenic diseases, like type 2 diabetes and obesity. The present study was conducted on Holstein calves to find the association between MOGAT1, MOGAT3/g.A229G, and MOGAT3/g.G1627A and growth traits. The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method was performed for genotyping the MOGAT1, MOGAT3/g.A229G, and MOGAT3/g.G1627A genes' locus using the TaqI, MspI, and BsuRI restriction enzyme. The allele frequency of A and G of the MOGAT1 locus was 0.79 and 0.21, respectively, while the genotype frequency was 0.65, 0.28, and 0.07 for AA, AG, and GG, respectively. While the allele and genotype frequencies of the MOGAT3/g.A229G locus were 00.57(A1), 0.43(G1), 0.35(A1A1), 0.45(A1G1), and 0.20(G1G1), the allele and genotype frequencies of the MOGAT3/g.G1627A locus were 0.49(A2), 0.51(G2), 0.25(A2A2), 0.49(A2G2), and 0.26(G2G2). Chi-square analysis showed that MOGAT3/g.G1627A distribution was at the Hardy–Weinberg disequilibrium (p < 0.05), and MOGAT1 and MOGAT3/g.A229G distribution was at the Hardy–Weinberg equilibrium (p > 0.05). In total, two statistical methods (general linear model (GLM) and PROC MIXED) were used to identify an association between gene locus and growth traits. An association analysis showed a statistically significant difference between the MOGAT1 and body weight, body length, and chest circumference, MOGAT3/g.A229G with average daily gain (ADG) and withers height, and MOGAT3/g.G1627A with body weight and body length (p < 0.05). The results confirmed that the MOGAT1, MOGAT3/g.A229G, and MOGAT3/g.G1627A locus are strong candidate genes that could be considered molecular markers for growth traits in cattle breeding.
Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the last reaction in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG). DGAT activity resides mainly in membrane-bound DGAT1 and DGAT2 in eukaryotes and bifunctional wax ester synthase-diacylglycerol acyltransferase (WSD) in bacteria, which are all membrane-bound proteins but exhibit no sequence homology to each other. Recent studies also identified other DGAT enzymes such as the soluble DGAT3 and diacylglycerol acetyltransferase (EaDAcT), as well as enzymes with DGAT activities including defective in cuticular ridges (DCR) and steryl and phytyl ester synthases (PESs). This review comprehensively discusses research advances on DGATs in prokaryotes and eukaryotes with a focus on their biochemical properties, physiological roles, and biotechnological and therapeutic applications. The review begins with a discussion of DGAT assay methods, followed by a systematic discussion of TAG biosynthesis and the properties and physiological role of DGATs. Thereafter, the review discusses the three-dimensional structure and insights into mechanism of action of human DGAT1, and the modeled DGAT1 from Brassica napus. The review then examines metabolic engineering strategies involving manipulation of DGAT, followed by a discussion of its therapeutic applications. DGAT in relation to improvement of livestock traits is also discussed along with DGATs in various other eukaryotic organisms.
Triacylglycerols are a major source of stored energy that are obtained either from the diet or can be synthesized to some extent by most tissues. Alterations in pathways of triacylglycerol metabolism can result in their excessive accumulation leading to obesity, insulin resistance, cardiovascular disease and nonalcoholic fatty liver disease. Most tissues in mammals synthesize triacylglycerols via the glycerol 3-phosphate pathway. However, in the small intestine the monoacylglycerol acyltransferase pathway is the predominant pathway for triacylglycerol biosynthesis where it participates in the absorption of dietary triacylglycerol. In this review, the enzymes that are part of both the glycerol 3-phosphate and monoacylglycerol acyltransferase pathways and their contributions to intestinal triacylglycerol metabolism are reviewed. The potential of some of the enzymes involved in triacylglycerol synthesis in the small intestine as possible therapeutic targets for treating metabolic disorders associated with elevated triacylglycerol is briefly discussed.
Although the general structure of the barrier between the gut and the blood is well known, many details are still missing. Here, we analyse the literature and our own data related to lipid transcytosis through adult mammalian enterocytes, and their absorption into lymph at the tissue level of the intestine. After starvation, the Golgi complex (GC) of enterocytes is in a resting state. The addition of lipids in the form of chyme leads to the initial appearance of pre-chylomicrons (ChMs) in the tubules of the smooth endoplasmic reticulum, which are attached at the basolateral plasma membrane, immediately below the ‘belt’ of the adhesive junctions. Then pre-ChMs move into the cisternae of the rough endoplasmic reticulum and then into the expansion of the perforated Golgi cisternae. Next, they pass through the GC, and are concentrated in the distensions of the perforated cisternae on the trans-side of the GC. The arrival of pre-ChMs at the GC leads to the transition of the GC to a state of active transport, with formation of intercisternal connections, attachment of cis-most and trans-most perforated cisternae to the medial Golgi cisternae, and disappearance of COPI vesicles. Post-Golgi carriers then deliver ChMs to the basolateral plasma membrane, fuse with it, and secret ChMs into the intercellular space between enterocytes at the level of their interdigitating contacts. Finally, ChMs are squeezed out into the interstitium through pores in the basal membrane, most likely due to the function of the actin-myosin ‘cuff’ around the interdigitating contacts. These pores appear to be formed by protrusions of the dendritic cells and the enterocytes per se. ChMs are absorbed from the interstitium into the lymphatic capillaries through the special oblique contacts between endothelial cells, which function as valves through the contraction-relaxation of bundles of smooth muscle cells in the interstitium. Lipid overloading of enterocytes results in accumulation of cytoplasmic lipid droplets, an increase in diameter of ChMs, inhibition of intra-Golgi transport, and fusion of ChMs in the interstitium. Here, we summarise and analyse recent findings, and discuss their functional implications.
Background: Dietary fat absorption involves the re esterification of digested triacylglycerol in the enterocytes, it is a biological process catalyzed by monoacylglycerol O acyltransferase 2 (MOGAT2, aka MGAT2), which is highly expressed in the small intestine. A previous study showed that the loss of the Mogat2 gene can prevent high fat diet induced obesity in mice. Obesity is associated with an increased risk of several types of cancer including postmenopausal breast cancer.
Methods: We collected 147 patients with triple negative breast adenocarcinoma to explore the relationship between the expression of MOGAT2 and patient overall survival. And we generated a Mogat2 deficient mouse mammary tumor model by crossing Mogat2 deficient mice with MMTV PyMT mice to examine the effect of losing MOGAT2 in vivo.
Results: Our founding suggest that obesity was induced by a relatively high fat diet (37% of calories from fat) in the mice with or without Mogat2 knockout. Mammary tumor development was deteriorated by a relatively high fat diet regardless of Mogat2 deficiency. As a compensation mechanism, upregulation of diacylglycerol O acyltransferases 1 and 2 (Dgat1 and Dgat2) in the Mogat2 deficient mice was found.
Conclusions: Elevated expression of MOGAT2 in triple negative breast adenocarcinoma predicts poorer patient overall survival. With the compensation of Dgat1 and Dgat2, Mogat2 deficiency alone cannot prevent fat diet induced obesity, nor prevent mammary tumor development in a mouse model.
The Arctic fox (Vulpes lagopus) is the only fox species occurring in the Arctic and has adapted to its extreme climatic conditions. Currently, the molecular basis of its adaptation to the extreme climate has not been characterized. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first V. lagopus genome assembly, which is assembled into chromosome fragments. The genome assembly has a total length of 2.345 Gb with a contig N50 of 31.848 Mb and a scaffold N50 of 131.537 Mb, consisting of 25 pseudochromosomal scaffolds. The V. lagopus genome had approximately 32.33% repeat sequences. In total, 21,278 protein‐coding genes were predicted, of which 99.14% were functionally annotated. Compared with 12 other mammals, V. lagopus was most closely related to V. Vulpes with an estimated divergence time of ~7.1 million years ago. The expanded gene families and positively selected genes potentially play roles in the adaptation of V. lagopus to Arctic extreme environment. This high‐quality assembled genome will not only promote future studies of genetic diversity and evolution in foxes and other canids but also provide important resources for conservation of Arctic species.
The dietary triacylglycerol (TAG) gets absorbed and accumulated in the body through the monoacylglycerol (MAG) pathway, which plays a major role in obesity and related disorders. The main enzyme of this pathway, monoacylglycerol acyltransferase 2 (MGAT2), is considered as a potential target for developing antiobesity compounds. Hence, there is a need for in vitro cell-based assays for screening the potential leads for MGAT2 inhibitors. Because of synthetic inhibitor’s side effects, there is an increased interest in natural extracts as potential leads. Hence, we have optimized a 2-MAG-induced TAG accumulation inhibitory cell-based assay to screen natural extracts using the HIEC-6 cell line. A concentration-dependent TAG accumulation was observed when the HIEC-6 cells were fed with exogenous 2-MAG. The TAG accumulation was confirmed by in situ BODIPY staining and was quantified. However, no TAG accumulation was seen when the cells were fed with exogenous DAG or TAG, suggesting MGAT2-mediated MAG uptake and its conversion to TAG. We demonstrated the utility of this assay by screening five different plant-based aqueous extracts. These extracts showed various inhibition levels (25% to 30%) of 2-MAG-induced TAG accumulation in the HIEC-6. The MGAT2 inhibitory potential of these extracts was confirmed by an in vitro MGAT2 assay. This cell-based assay adds a new methodology for screening, developing, and evaluating MGAT2 inhibitors for addressing obesity and related disorders.
Single nucleotide polymorphism (SNP) has recently become one of the ideal genetic markers. SNP refers to the DNA sequence polymorphism caused by double nucleotide variation in the genome, including the conversion or transversion of segmented bases. The synthesis and metabolism of triglycerides are related to the changes of energy in the body of livestock, which in turn affects their growth and development. Studies have shown that MOGAT1 gene plays a role in the route of triglyceride synthesis. PCR-RFLP and agarose gel electrophoresis technology were used to type the SNP site of MOGAT1 gene at g.25940T > C in this study. Association analysis between typing results and growth trait data was detected by SPSS 20.0 software. Results show that MOGAT1 gene was in a low level of heterozygosity in Xianan, Qinchuan and Pinan cattle population (0 < PIC < 0.25), and in middle level of heterozygosity in YL cattle population(0.25 < PIC < 0.5). And genotype 'AA' was dominant gene in Chinese cattle population. In QC and XN cattle, genotype of GG possess advantage on Body weight (P < 0.05); in YL cattle, individuals with genotype of homozygous mutation decreased significantly on Chest depth (P < 0.05). The purpose of this research is to provide theoretical materials for molecular breeding of yellow cattle and to promote the process of improving the growth traits of Chinese local yellow cattle.
MicroRNA-221-3p (miR-221-3p) is associated with both metabolic diseases and cancers. However, its role in terminal adipocyte differentiation and lipid metabolism are uncharacterized. miR-221-3p or its inhibitor were transfected into differentiating or mature human adipocytes. Triglyceride (TG) content and adipogenic gene expression were monitored, global lipidome analysis was carried out, and mechanisms underlying the effects of miR-221-3p were investigated. Finally, cross-talk between miR-221-3p expressing adipocytes and MCF-7 breast carcinoma (BC) cells was studied, and miR-221-3p expression in tumor-proximal adipose biopsies from BC patients analyzed. miR-221-3p overexpression inhibited terminal differentiation of adipocytes, as judged from reduced TG storage and gene expression of the adipogenic markers SCD1, GLUT4, FAS, DGAT1/2, AP2, ATGL and AdipoQ, whereas the miR-221-3p inhibitor increased TG storage. Knockdown of the predicted miR-221-3p target, 14–3-3γ, had similar antiadipogenic effects as miR-221-3p overexpression, indicating it as a potential mediator of mir-221-3p function. Importantly, miR-221-3p overexpression inhibited de novo lipogenesis but increased the concentrations of ceramides and sphingomyelins, while reducing diacylglycerols, concomitant with suppression of sphingomyelin phosphodiesterase, ATP citrate lyase, and acid ceramidase. miR-221-3p expression was elevated in tumor proximal adipose tissue from patients with invasive BC. Conditioned medium of miR-221-3p overexpressing adipocytes stimulated the invasion and proliferation of BC cells, while medium of the BC cells enhanced miR-221-3p expression in adipocytes. Elevated miR-221-3p impairs adipocyte lipid storage and differentiation, and modifies their ceramide, sphingomyelin, and diacylglycerol content. These alterations are relevant for metabolic diseases but may also affect cancer progression.
The current understanding of the genetic architecture of lipids has largely come from genome-wide association studies (GWAS). To date, few GWAS have examined the genetic architecture of lipids in Polynesians, and none have in Samoans, whose unique population history, including many population bottlenecks, may provide insight into the biological foundations of variation in lipid levels. Here we performed a GWAS of four fasting serum lipid levels: total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglycerides (TG) in a sample of 2849 Samoans, with validation genotyping for associations in a replication cohort comprising 1798 Samoans and American Samoans. We identified multiple genome-wide significant associations (P < 5 × 10⁻⁸) previously seen in other populations—APOA1 with TG, CETP with HDL, and APOE with TC and LDL—and several suggestive associations (P < 1 × 10⁻⁵), including an association of variants downstream of MGAT1 and RAB21 with HDL. However, we observed different association signals for variants near APOE than what has been previously reported in non-Polynesian populations. The association with several known lipid loci combined with the newly identified associations with variants near MGAT1 and RAB21 suggest that while some of the genetic architecture of lipids is shared between Samoans and other populations, part of the genetic architecture may be Polynesian-specific.
Diacylglycerol acyltransferase2 (DGAT2) catalyzes the final step in triacylglycerol (TAG) synthesis. Genetic knockdown or pharmacological inhibition of DGAT2 leads to reduction in very-low-density lipoprotein (VLDL) TAG secretion and hepatic lipid levels in rodents, indicating DGAT2 may represent an attractive therapeutic target for treatment of hyperlipidemia and hepatic steatosis. We have previously described potent and selective imidazopyridine DGAT2 inhibitors with high oral bioavailability. However, the detailed mechanism of DGAT2 inhibition has not been reported. Herein, we describe imidazopyridines represented by (PF-06424439, 1) and (2) as long residence time inhibitors of DGAT2. We demonstrate that 1 and 2 are slowly reversible, time-dependent inhibitors, which inhibit DGAT2 in a noncompetitive mode with respect to the acyl-CoA substrate. Detailed kinetic analysis demonstrated that 1 and 2 inhibit DGAT2 in a two-step binding mechanism, in which the initial enzyme-inhibitor complex (EI) undergoes an isomerization step resulting in a much higher affinity complex (EI*) with overall inhibition constants (Ki* values) of 16.7 and 16.0 nM for 1 and 2, respectively. The EI* complex dissociates with dissociation half-lives of 1.2 and 1.0 hr for 1 and 2, respectively. A binding assay utilizing [125I]-labeled imidazopyridine demonstrated that imidazopyridine binding to DGAT2 mutant enzymes, H161A and H163A, dramatically decreased to 11-17% of that of the WT enzyme, indicating that these residues are critical for imidazopyridines to bind to DGAT2. Taken together, imidazopyridines may thus represent a promising lead series for the development of DGAT2 inhibitors that display an unprecedented combination of potency, selectivity, and in vivo efficacy.
Background/aims:
Aberrant activation of the Wnt/β-catenin signaling, which arises from the accumulation of mutant β-catenin in the cell, is one of the most common driving forces in hepatocellular carcinoma (HCC). We previously identified several genes that are regulated on the overexpression of β-catenin in the HCC cell line that are suggested to be novel Wnt/β-catenin targets playing effective roles in cancer. The aim of the present study was to elucidate the roles of these putative target genes in tumorigenesis with an in vivo analysis in Drosophila.
Materials and methods:
We selected 15 genes downregulated in two Drosophila cancer models.
Results:
The results from the RNAi mini-screen revealed novel roles for the analyzed putative Wnt/β-catenin target genes in tumorigenesis. The downregulation of the analyzed nine genes led to tumor formation as well as metastasis in Drosophila, suggesting a tumor suppressor function. On the other hand, the knockdown of the other two genes suppressed tumor and metastasis formations and disturbed the development of the analyzed eye tissues, indicating an oncogenic or developmental role for these genes.
Conclusion:
These findings could serve to identify novel subjects for cancer research in order to provide insight into the diagnostic and therapeutic processes of several cancer types.
The esterification of a fatty acyl moiety to diacylglycerol to form triacylglycerol (TAG) is catalysed by two diacylglycerol O-acyltransferases (DGATs) encoded by genes belonging to two distinct gene families. The enzymes are referred to as DGAT1 and DGAT2 in order of their identification. Both proteins are transmembrane proteins localized in the endoplasmic reticulum. Their membrane topologies are however significantly different. This difference is hypothesized to give the two isozymes different abilities to interact with other proteins and organelles and access to different pools of fatty acids, thereby creating a distinction between the enzymes in terms of their role and contribution to lipid metabolism. DGAT1 is proposed to have dual topology contributing to TAG synthesis on both sides of the ER membrane and esterifying only the pre-formed fatty acids. There is evidence to suggest that DGAT2 translocates to the lipid droplet (LD) and associates with other proteins, and synthesizes cytosolic and luminal apolipoprotein B associated LD-TAG from both endogenous and exogenous fatty acids. The aim of this review is to differentiate between the two DGAT enzymes by comparing the genes that encode them, their proposed topologies, the proteins they interact with, and their roles in lipid metabolism.
Monoacylglycerol transferase 2 (MGAT2) is a pivotal enzyme in the monoacylglycerol pathway for triacylglycerol synthesis. The pathway for triacylglycerol synthesis has provided several attractive targets for drug discovery in the treatment of metabolic diseases. Marketed drugs that inhibit enzymes in this pathway include orlistat (pancreatic lipase inhibitor), lomitapide (mitochondrial transfer protein inhibitor), and mipomersen (apolipoprotein B synthesis inhibitor) but poor gastrointestinal (GI) tolerability or safety considerations have limited their use and indications. In addition, several inhibitors of diacylglycerol transferase 1 (DGAT1) have advanced to the clinic but were withdrawn due to poor GI tolerability. This perspective first discusses the biological rationale in support of inhibition of MGAT2 as a therapeutic approach that may offer a distinct and superior efficacy versus GI tolerability profile, and then reviews advances in the discovery of small molecule MGAT2 inhibitors for the treatment of metabolic diseases and non-alcoholic steatohepatitis (NASH).
Intestinal lipid absorption is an efficient and highly complex process. Here we review the main pathways involved in intestinal processing of dietary lipid. We discuss the regulation of enterocyte fatty acid transport and the functions in absorption of lipid transporters, scavenger receptor B1 (SR-B1), CD36 or SR-B2, and fatty acid transport protein 4 (FATP4). We describe recent findings highlighting importance of the remodeling of membrane phospholipids in particular by lysophosphatidylcholine acyltransferase 3 (LPCAT3), in facilitating passive diffusion of fatty acid and promoting triacylglycerol (TAG) assembly into lipoproteins. We review what we currently know about the most prominent intestinal enzymes involved in TAG synthesis in the endoplasmic reticulum including long-chain acyl-CoA synthetase (ACSL), monoacylglycerol acyltransferase (MGAT), and diacylglycerol acyltransferase (DGAT). We also provide a detailed and updated review on the mechanisms and regulation of prechylomicron formation, assembly, and transport from the endoplasmic reticulum to the Golgi for chylomicron generation, with special reference to the role of CD36 and liver fatty acid-binding protein (LFABP).
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.
The activities of the proposed triacylglycerol synthetase complex, acyl-CoA ligase, acyl-CoA acyltransferase (AAT), monoacylglycerol
acyltransferase (MGAT), and diacylglycerol acyltransferase (DGAT), coeluted upon Cibacron blue 3GA-agarose affinity chromatography
of detergent-solubilized rat intestinal microsomes. The AAT activity is associated with a 54-kDa protein, that binds covalently
an acyl group from acyl-CoA via a thiol ester linkage (Lehner, R. and Kuksis, A.(1993) J. Biol. Chem. 268, 24726-24733). Reagents that prevent the acyl-AAT formation inhibit triacylglycerol synthesis as does the removal of
AAT from the complex by immunoprecipitation. In the absence of mono- and diacylglycerol acceptors, the acyl group is transferred
to water. It is proposed that triacylglycerol synthesis proceeds via a sequential transfer of acyl groups from acyl-CoA ligase
to the AAT, from which they are passed to the mono- and diacylglycerol acyltransferases for incorporation into the di- and
triacylglycerols depending on the availability of the acyl acceptors.
Studies involving the cloning and disruption of the gene for acyl-CoA:diacylglycerol acyltransferase (DGAT) have shown that
alternative mechanisms exist for triglyceride synthesis. In this study, we cloned and characterized a second mammalian DGAT,
DGAT2, which was identified by its homology to a DGAT in the fungus Mortierella rammaniana. DGAT2 is a member of a gene family that has no homology with DGAT1 and includes several mouse and human homologues that
are candidates for additional DGAT genes. The expression of DGAT2 in insect cells stimulated triglyceride synthesis 6-fold
in assays with cellular membranes, and DGAT2 activity was dependent on the presence of fatty acyl-CoA and diacylglycerol,
indicating that this protein is a DGAT. Activity was not observed for acyl acceptors other than diacylglycerol. DGAT2 activity
was inhibited by a high concentration (100 mm) of MgCl2 in anin vitro assay, a characteristic that distinguishes DGAT2 from DGAT1. DGAT2 is expressed in many tissues with high expression levels
in the liver and white adipose tissue, suggesting that it may play a significant role in mammalian triglyceride metabolism.
The monoglycerol acyltransferase (EC 2.3.1.22) (recommended name acylglycerol palmitoltransferase) activities from rat intestinal mucosa and suckling liver microsomes were compared in order to determine why substrate specificities differed in the two tissues. Suckling liver monoacylglycerol acyltransferase activity was highly specific for sn-2-mono-C18:1 glycerol and acylated rac-1-mono-C18:1 glycerol and 1- and 2-mono-C18:1 glycerol ethers poorly. In contrast, the substrate specificity of intestinal monoacylglycerol acyltransferase activity was broad. 1-Acyl- and 1- and 2-alkylglycerols were acylated at rates that were 45-78% of the rate observed with the preferred substrate sn-2-mono-C18:1 glycerol. Partial heat inactivation did not alter these relative specific activities, making it unlikely that intestinal microsomes contained a second acyltransferase capable of acylating the alternate substrates. The hypothesis that intestine and liver contain non-identical monoacylglycerol acyltransferase activities was further tested. Intestinal mucosa monoacylglycerol acyltransferase was much more thermolabile than the liver activity. Incubation with 50 microM diethylpyrocarbonate inactivated liver monoacylglycerol acyltransferase activity 84% but had little effect on the intestinal activity. Hydroxylamine completely reversed diethylpyrocarbonate inactivation, suggesting that critical histidine residues were more accessible in liver monoacylglycerol acyltransferase. 2,4,6-Trinitrobenzene sulfonic acid inactivated hepatic monoacylglycerol acyltransferase more than the intestinal activity, suggesting that critical lysine residues were more accessible. The intestinal and liver activities were also differently affected by acetone, detergents, MgCl2, phospholipids, and bovine serum albumin. Taken as a whole, the data strongly suggest that rat intestinal mucosa and suckling liver contain tissue-specific monoacylglycerol acyltransferase isoenzymes.
Hepatic monoacylglycerol acyltransferase activity is 700-fold higher during the suckling period than in the adult rat. Specific activity in total particulate preparations rose from 9.4 nmol/min/mg before birth to a peak of 78 nmol/min/mg on the 6th to 8th postnatal days. Monoacylglycerol acyltransferase activity fell sharply after day 8 and was 1.6 and 0.1 nmol/min/mg on day 28 and in adult rats, respectively. The activity had a pH optimum at 8.0 and was activated by albumin and by phospholipids. With [3H]palmitoyl-CoA and sn-2-monooleoylglycerol, more than 96% of the products were di- and triacylglycerols. More than 92% of the diacylglycerol product was the 1,2 isomer. The activity was stable at 43 degrees C for 50 min. Thermal inactivation showed t 1/2 values of 8 min and 4.5 min at 53.5 and 55 degrees C, respectively. In suckling rats, monoacylglycerol acyltransferase activities in liver and intestinal mucosa were 150- to 800-fold higher than in other tissues. Monoacylglycerol acyltransferase activity was 12.5-fold greater with palmitoyl-CoA than with octanoyl-CoA. Acetyl-CoA was not a substrate. The sn-2-monoacylglycerols were strongly preferred over the sn-1 isomers. No direct relationship was noted between 2-monoacylglycerol chain length and apparent Km value. The presence of high levels of monoacylglycerol acyltransferase activity in suckling rat liver suggests that the monoacylglycerol pathway functions as a major route of hepatic glycerolipid synthesis during the suckling period but not in adult animals.
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.
Biosynthesis of diacylglycerols in plants occurs mainly through the acylation of lysophosphatidic acid in the microsomal membranes. Here we describe the first identification of diacylglycerol biosynthetic activity in the soluble fraction of developing oilseeds. This activity was NaF-insensitive and acyl-CoA-dependent. Diacylglycerol formation was catalyzed by monoacylglycerol (MAG) acyltransferase (EC ) that transferred an acyl moiety from acyl-CoA to MAG. The enzyme was purified by successive chromatographic separations on octyl-Sepharose, blue-Sepharose, Superdex-75, and palmitoyl-CoA-agarose to apparent homogeneity from developing peanut (Arachis hypogaea) cotyledons. The enzyme was purified to 6,608-fold with the final specific activity of 15.86 nmol min(-1) mg(-1). The purified enzyme was electrophoretically homogeneous, and its molecular mass was 43,000 daltons. The purified MAG acyltransferase was specific for MAG and did not utilize any other acyl acceptor such as glycerol, glycerol-3-phosphate, lysophosphatidic acid, and lysophosphatidylcholine. The K(m) values for 1-palmitoylglycerol and 1-oleoylglycerol were 16.39 and 5.65 micrometer, respectively. The K(m) values for 2-monoacylglycerols were 2- to 4-fold higher than that of the corresponding 1-monoacylglycerol. The apparent K(m) values for palmitoyl-, stearoyl-, and oleoyl-CoAs were 17.54, 25.66, and 9.35 micrometer, respectively. Fatty acids, phospholipids, and sphingosine at low concentrations stimulated the enzyme activity. The identification of MAG acyltransferase in oilseeds suggests the presence of a regulatory link between signal transduction and synthesis of complex lipids in plants.
Acyl CoA:diacylgycerol acyltransferase (EC2.3.1.20; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity,
were isolated from the lipid bodies of the oleaginous fungusMortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular
masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based
on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences
encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes
isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified
DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other
gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals,
which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer
to the M. ramanniana genes asMrDGAT2A and MrDGAT2B.
Traumatic brain injury triggers the accumulation of harmful mediators that may lead to secondary damage. Protective mechanisms to attenuate damage are also set in motion. 2-Arachidonoyl glycerol (2-AG) is an endogenous cannabinoid, identified both in the periphery and in the brain, but its physiological roles have been only partially clarified. Here we show that, after injury to the mouse brain, 2-AG may have a neuroprotective role in which the cannabinoid system is involved. After closed head injury (CHI) in mice, the level of endogenous 2-AG was significantly elevated. We administered synthetic 2-AG to mice after CHI and found significant reduction of brain oedema, better clinical recovery, reduced infarct volume and reduced hippocampal cell death compared with controls. When 2-AG was administered together with additional inactive 2-acyl-glycerols that are normally present in the brain, functional recovery was significantly enhanced. The beneficial effect of 2-AG was dose-dependently attenuated by SR-141761A, an antagonist of the CB1 cannabinoid receptor.
A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels.
This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular
events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids,
particularly choline phospholipids, by phospholipase D and phospholipase A2 may also take part in cell signaling. The products
of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation
of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.
Previous studies by Coleman and Haynes (1986, J. Biol. Chem. 261, 224-228) indicated the presence of two different tissue specific monoacylglycerol acyltransferases (MGAT) in intestinal mucosa and suckling rat liver. The evidence was based upon the differential responses of these two isoenzymes to various treatments including, the effects of temperature, proteolysis, protein modification reagents, detergents, and divalent cations. In the present investigation, we have used some of these criteria to determine the identity of adipose enzyme with the MGAT present in liver and intestinal microsomes. The properties of adipose and intestinal enzymes were similar in several respects, but differed from the liver enzyme. This suggests the possibility that MGAT activities from adipose and intestine may be mediated by the same enzyme protein, whereas the liver MGAT may be a separate isoenzyme as proposed earlier. The most distinguishing feature of the liver enzyme was its ability to sustain both high temperature (52 degrees C) and resist proteolysis. However, when disrupted microsomal preparations were used, the liver enzyme was both thermolabile and protease sensitive, as observed for the intestinal and adipose MGAT. Possibly, the location of liver MGAT within the membranes may be responsible for such unique properties of liver MGAT.
We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol-3-phosphate.
Acyl-CoA: monoglyceride acyltransferase (MGAT; EC 2.3.1.22) has been studied in human small intestinal mucosa by means of a spectrophotometric method based on the detection of liberated CoA employing 5,5'-dithiobis-(2-nitrobenzoic acid). With optimal assay conditions available the pH optimum was spread between 7.0 and 7.7 with a maximum at a pH of 7.4. Dependent on its concentration one of the substrates, palmitoyl-CoA, caused severe inhibition which was largely prevented by the addition of albumin. Using palmitoyl-CoA and 1-monooleoylglycerol as substrates specific activities were 23.0 +/- 8.4 in total homogenates compared with 92.9 +/- 28.3 nmol CoA released/min/mg protein in microsomal fractions from jejunal mucosa. Concerning the substrate specificity, a broad acyl-donor pattern exists with maximal activities for C10:0 to C16:0, the highest for C16:0. A preferential esterification of acyl-acceptors was shown for 2-monoacylglycerols as compared with the 1-isomers, and for monoacylglycerols with unsaturated versus saturated fatty acids. MGAT was mainly localized in the microsomal fraction. The properties of MGAT from human small intestine are discussed with respect to the intestinal enzyme from other species.
The activities of the proposed triacylglycerol synthetase complex, acyl-CoA ligase, acyl-CoA acyltransferase (AAT), monoacylglycerol acyltransferase (MGAT), and diacylglycerol acyltransferase (DGAT), coeluted upon Cibacron blue 3GA-agarose affinity chromatography of detergent-solubilized rat intestinal microsomes. The AAT activity is associated with a 54-kDa protein, that binds covalently an acyl group from acyl-CoA via a thiol ester linkage (Lehner, R. and Kuksis, A. (1993) J. Biol. Chem. 268, 24726-24733). Reagents that prevent the acyl-AAT formation inhibit triacylglycerol synthesis as does the removal of AAT from the complex by immunoprecipitation. In the absence of mono- and diacylglycerol acceptors, the acyl group is transferred to water. It is proposed that triacylglycerol synthesis proceeds via a sequential transfer of acyl groups from acyl-CoA ligase to the AAT, from which they are passed to the mono- and diacylglycerol acyltransferases for incorporation into the di- and triacylglycerols depending on the availability of the acyl acceptors.
Although migrating birds use stored triacylglycerol as their primary fuel for flight, they must retain sufficient stores of omega 6 and omega 3 fatty acids to sustain reproduction after the spring migration. Hepatic monoacylglycerol:acyl-coenzyme A acyltransferase (EC 2.3.1.22) (MGAT) activity is associated with physiological periods in which lipolysis and beta-oxidation are prominent, and it may also play a role in the selective retention of certain essential fatty acids. Therefore, we characterized MGAT activity in adipose tissue from the white-throated sparrow (Zonotrichia albicollis), a migratory bird. MGAT specific activity from adipose tissue and liver, respectively, was 22.2 +/- 7.27 and 0.79 +/- 0.35 nmol/min/mg of total particulate protein. Activity did not vary seasonally or between male and female birds. Specific activity increased 4.3-fold in the presence of 75 micrograms of phosphatidylcholine and phosphatidylserine (1:1, w/w). MGAT acylated sn-1(3)-monooleoylglycerol, sn-2-monooleylglycerol ether and sn-1(3)-monooleylglycerol ether at 7.5, 5.7 and 1.7%, respectively, of the rate observed with sn-2-monooleoylglycerol. An initial lag phase observed at low concentrations of palmitoyl-CoA was corrected by adding 2 mM MgCl2, Mg(NO3)2 or CaCl2, suggesting a requirement for divalent cations. MGAT acylated sn-2-monolinolenoylglycerol and sn-2-monolinoleoylglycerol in preference to sn-2-monooleoylglycerol. Specificity of MGAT for sn-2-monoacylglycerols and the probable enhanced affinity fo sn-2-monoacylglycerols of specific acyl chains may allow selected omega 6 and omega 3 fatty acids to be retained within the adipocyte, while nonessential fatty acids are released for beta-oxidation in flight muscles.
In the suckling rat, chick embryo, and hibernating marmot, fatty acids provide the major source of energy, and despite the high rate of hepatic beta-oxidation, these animals selectively retain long-chain polyunsaturated derivatives of C18:2n-6 and C18:3n-3. To determine whether the hepatic microsomal activity monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) could provide a mechanism to selectively acylate monoacylglycerols that contain essential fatty acids, we tested the ability of MGAT activity from each of the three species to acylate sn-2-monoC18:1-, sn-2-monoC18:2-, sn-2-monoC18:3-, and sn-2-monoC20:4-glycerols. Hepatic MGAT activity acylated sn-2-monoC18:3-glycerol and sn-2-monoC18:2-glycerol in preference to sn-2-monoC18:1-glycerol in each of the three different lipolytic animals. MGAT's acyl group specificity could not be explained by invoking differences in membrane fluidity because the apparent affinity for sn-2-monoC20:4-glycerol was not increased. Further, sn-2-monoC18:3-glycerol remained a preferred substrate under assay conditions when both the C18:3 and C18:1 species were present in equal amounts. As would be predicted in the presence of high activity of a selective MGAT, the hepatic glycerolipids from neonatal rats showed increases in dienoic, trienoic, and C22:6 fatty acids and relative decreases in monoenoic, saturated, and C20:4 fatty acids. We hypothesize that, during lipolysis, the reacylation of sn-2-monoacylglycerols by MGAT may provide a mechanism by which essential fatty acids are retained within specific tissues.
Rat hepatic microsomal monoacylglycerol acyltransferase (MGAT) is a developmentally expressed enzyme that catalyzes the formation of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and fatty acyl-CoA. Treatment of suckling rat liver microsomes with various detergents showed that 0.3% Triton X-100, a nonionic detergent, solubilized a maximum amount of both protein (66%) and MGAT activity (56%). After solubilization with Triton X-100, MGAT was then purified 205-fold by sequential chromatography on QAE-Sephadex, CM-Sepharose (Fast Flow), and hydroxylapatite. Addition of phospholipids to the reaction mixture stimulated the purified enzyme activity more than 1.8-fold. sn-1,2-DiC18: 1-glycerol activated purified MGAT activity. Purified MGAT activity was specific for sn-2-monoacylglycerol; the activity with rac-1-monoC18:1-glycerol and rac-1- and sn-2-monoC18:1-glycerol ethers was less than 4% of the activity with sn-2-monoC18:1-glycerol. The purified MGAT had an isoelectric point of 9.7. The apparent Km and Vmax values of the purified enzyme for sn-2-monoC18:1-glycerol were 21 microM and 1036 nmol/min/mg, respectively. The apparent Km value for palmitoyl-CoA was 6.5 microM. Purified MGAT activity acylated sn-2-monoC18:2-glycerol and sn-2-monoC18:3-glycerol in preference to sn-2-monoC18:1-glycerol, consistent with a role for the monoacylglycerol pathway in retaining essential fatty acids.
Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol synthetase of rat intestinal mucosa. With 2-oleoyl[-3H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97-98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was the sn-1,2- and 10% the sn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was the sn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-14C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-14C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-3H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.
Protein kinase C (PKC), a family of related serine-threonine kinases, is a key player in the cellular responses mediated by the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. The traditional view of PKCs as DAG/phospholipid-regulated proteins has expanded in the last few years by three seminal discoveries. First, PKC activity and maturation is controlled by autophosphorylation and transphosphorylation mechanisms, which includes phosphorylation of PKC isozymes by phosphoinositide-dependent protein kinases (PDKs) and tyrosine kinases. Second, PKC activity and localization are regulated by direct interaction with different types of interacting proteins. Protein-protein interactions are now recognized as important mechanisms that target individual PKCs to different intracellular compartments and confer selectivity by associating individual isozymes with specific substrates. Last, the discovery of novel phorbol ester receptors lacking kinase activity allows us to speculate that some of the biological responses elicited by phorbol esters or by activation of receptors coupled to elevation in DAG levels could be mediated by PKC-independent pathways.
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