DGAT1 Presentation PDF

DGAT1 Is Not Essential for Intestinal Triacylglycerol Absorption or Chylomicron Synthesis Janet McDermott, Grace Lalanne, Zack Douchy, Jack Amos, Jane Nguyen A quick review… Triacylglycerol (TAG, triglyceride): glycerol backbone Free fatty acids: with 3 fatty acid tails...

DGAT1 Is Not Essential for Intestinal Triacylglycerol Absorption or Chylomicron Synthesis Janet McDermott, Grace Lalanne, Zack Douchy, Jack Amos, Jane Nguyen A quick review… Triacylglycerol (TAG, triglyceride): glycerol backbone Free fatty acids: with 3 fatty acid tails broken off of TAGS and diacylglycerol (DAGS) Monoacylglycerol (MAG): glycerol backbone with 1 fatty acid tail A quick review… Chylomicrons are the largest and least dense class of lipoproteins, and they’re composed of roughly 90% TAGs. In order for chylomicrons to be synthesized, dietary fats must be broken down in the lumen before being resynthesized in the enterocytes of the small intestine. Newly synthesized TAGs are processed by the Golgi to assemble chylomicrons, which are then transported to the lacteals of the lymphatic system. Relevant enzymes and their reactions free Monoacylglycerol fatty acyltransferase (MGAT) free acid fatty acid Diacylglycerol acyltransferase (DGAT) 1 and 2 Both DGAT1 and DGAT2 isoforms are expressed in the small intestine Relevant enzymes and their reactions Diacylglycerol transacylase Diacylglycerol transacylase transfers an acyl group from one diacylglycerol to another, forming a triacylglycerol and a monoacylglycerol.  Knowledge Gap & Research Questions Researchers sought to better understand the relative contributions of DGAT1, DGAT2, and DAG transacylase in TAG synthesis and absorption. Dgat1-/- mice were utilized to further define the function of DGAT1 in the intestine. Research Questions: 1. Does DGAT1 deficiency alter TAG metabolism in enterocytes? 2. Can mice that lack DGAT1 synthesize chylomicron-sized lipoprotein particles? 3. What are the relative contributions of DGAT1, DGAT2, and DAG transacylase in intestinal TAG synthesis and absorption?  Figure 1 experimental methods Researchers isolated the small intestines of nonfasted WT mice, and divided into 5 equal sections. RNA from Northern blot intestinal mucosa was obtained for mRNA purification. analysis They used the pooled mRNA and radioactive P-labeled cDNA probes for Dgat1 and Dgat2 to generate the Northern blot analysis pictured in Figure 1A. Researchers performed in situ hybridization using fixed intestine sections from WT mice. The sections were hybridized with radioactive S-labeled Dgat1 RNA in situ probes. After, the sections were washed, dehydrated, hybridization dipped in photographic emulsion NTB₂, and developed 8 weeks later to produce Figure 1B. Figure 1: DGAT gene expression in mouse small intestine Since it is believed DGAT1 serves a role in TAG synthesis and absorption, if the levels of DGAT1 expression are analyzed in the intestine, then they would expect to see widespread expression and especially high expression in regions where lipid processing occurs. A. Dgat1 expressed in all segments with the highest expression in proximal intestine and lowest expression in most distal region. B. In situ hybridization shows high expression of Dgat1 mRNA along the tip of the villus.  Figure 2 experimental methods 1. 2. 4. 6. Fasted wild-type and Dgat1-/- Blood samples were obtained from Ultracentrifugation was used to isolate chylomicrons and mice were given a lipid bolus low-density lipoprotein (LDL) from plasma samples. Chylomicrons are quantified retro-orbital plexus 1hr after lipid using densitometry intragastrically by gavage. administration. 3. 5. 8. Plasma triacylglycerols quantities were measured with colorimetric assay Plasma lipoproteins were separated by agarose gel electrophoresis and stained for 7. lipid with Fat Red 7B Fasted mice were administered with retinol palmitate mixed with corn oil. This is the indicator of chylomicron synthesis The mice was fed with [125I]BMIPP, a 3-methyl-branched fatty acid analog slowly and secretion. Blood samples was obtained before and 2h after catabolized with by β-oxidation, in olive oil. This experiment measure how administration. Plasma retinol levels were analyzed with effective in absorbing BMIPP by mice intestine. Blood sample was obtained from reversed-phase liquid chromatography. the mice tail after overnight fasting. Gamma counter was used to measure [125I]BMIPP radioactivity.  Figure 2: Diminished chylomicronemia after an acute dietary lipid challenge A. Plasma triacylglycerols concentration was significantly higher in wild-type mice after intragastric oil bolus compared to Dgat1-/- mice. (n=11 per genotype). B. Visible chylomicrons appeared on top of the test tube in a wild-type plasma sample after ultracentrifugation while there was a small amount of chylomicrons observed in mutant plasma sample. (n=4 per genotype) C. Gel electrophoresis revealed chylomicrons at the origin, LDL in β band, VLDL in pre-β band, and HDL in ⍺ band. All lipoproteins present in both genotypes. D. Densitometry of the gel revealed chylomicrons concentration was roughly 60% lower in Dgat1-/- mice. E. Plasma levels of BMIPP were 75% lower in ➔ Conclusion: These results show that Dgat1-/- Dgat1-/- mice compared to the wild-types. mice have reduced postabsorptive chylomicronemia F. Plasma retinol level was 50% lower in Dgat1-/- in response to an acute lipid challenge. mice compared to wild-types. Figure 3 experimental methods Intestinal tissues from non fasted mice fed different diets were fixed for electron microscope analyzation. The researchers stained the tissue with Osmium tetroxide in a PBS. Sections of these were dehydrated in ethanol and then counterstained with toluidine blue. Then the tissue was subject to another Osmium Tetroxide procedure and further embedded on the analyzation plate for electron microscopy. Soon, ultrathin sections of these tissue were stained under a low concentration lead citrate solution for photograph under the electron microscope. Since TAG absorption and chylomicron synthesis is depleted, then lipids will proceed to accumulate in the cytoplasm of enterocytes. If Dgat1-/- mice are fed a diet high in fat, it would be expected to see lipid accumulation in fixed tissue. Figure 3: Accumulation of neutral lipid-staining droplets in the cytoplasm of enterocytes from Dgat1-/- mice fed a high fat diet for 3 weeks Researchers isolated the small intestines of nonfasted WT mice, and divided into 5 sections that were equal in length. Researchers utilized RNA STAT60 to extract the RNA from intestinal mucosa. Subsequently, equal amounts of RNA were obtained for mRNA purification. Lipid droplets did not accumulate in both types of mice given a diet of rodent chow. Not shown in the paper but important, DGAT-/- mice become overwhelmed when fed a diet high in fat content. Figure 4 experimental methods Since researchers determined that lipid droplets accumulated in the enterocytes of Dgat1-/- mice, they then aimed to determine the composition of these lipid droplets. They extracted lipids from the proximal sections of the small intestine of mice fed a high milk fat diet, and analyzed the composition using thin-layer chromatography (TLC). Intestinal segment #2 extracted from n=3 mice of each genotype Extract lipids from homogenate with methanol and chloroform solution Tissue homogenization Lipids are separated by TLC by size and polarity, allowing researchers to determine the composition of the lipids in the sample Figure 4: Accumulation of triacylglycerol and diacylglycerol in the small intestine of Dgat1-/- mice fed a high milk fat diet for 3 weeks Compared to WT mice, Dgat1-/- had significantly higher TAG and DAG levels in the enterocytes of the small intestine ○ Densitometry of the bands was used to quantify the amounts of TAGs and DAGs in WT compared to control The relative increase of TAGs was greater than for DAGs ○ Since DGAT1 was not present, then the compensatory mechanisms for TAG synthesis predominated. If diacylglycerol transacylase is used to synthesize TAGS, the DAG levels would decrease. Figure 5 and 6 Experimental Methods Both Figure 5 and 6 were imaged using electron microscopy. The tissues were stained for lipid by the imidazole-buffered osmium tetroxide procedure, stained en bloc (all together) in 2% aqueous uranyl acetate for 1 h at 4 °C, and embedded in Epon 812. Ultrathin sections were stained for 5 min with 0.8% lead citrate and photographed with an electron microscope. Figure 5 and 6 include an enterocyte from a wild type mouse (A) and a Dgat1-/- mice (B), both fed a high corn oil diet, which makes the lipid stain used more effective. Figure 5: Ultrastructural analysis of Dgat1-/- enterocytes Since we know an important step in triacylglycerol synthesis involves the presence of diacylglycerol acyltransferase (DGAT), if we remove Dgat1 (the gene that encodes for the DGAT1 enzyme), then we suspect changes in chylomicron synthesis. Purpose: determine if Dgat1-/- mice could synthesize chylomicron-sized particles to see if DGAT1 was needed for chylomicron synthesis. Figure 5, chylomicron-sized particles are present in both wild type (A) and Dgat1-/- mice (B) However, some enterocytes of the Dgat1-/- mice contained a single, large lipid droplet (LD) in the cytoplasm. Figure 6: Chylomicron-sized particles in Dgat1-/- enterocytes Figure 6 displays a more magnified image of the wild type (A) and Dgat1-/- mice (B) enterocytes, further showcasing the abundant presence of chylomicron-sized particles in: Green: membrane components of Golgi apparatus Orange: secretory vesicles Red: endoplasmic reticulum cisternae Both Figures 5 and 6 support the idea that DGAT1 is not essential for chylomicron synthesis.  Figure 7 experimental methods Activity of DGAT and DAG transacylase were measured from tissue homogenates. For DGAT assay, a mixture of DAG, oleoyl-CoA, and MgCl2 was incubated to allow the incorporation of oleoyl-CoA into TAGs. For DAG transacylase assay, a mixture of DAG, oleoyl-CoA, and MgCl2 was incubated to measure the incorporation of DAG into TAGs. After, lipids were extracted via chloroform:methanol, dried, and separated by TLC. The TAG bands were scraped from the plates and radioactivity was measured via scintillation counting. The incorporation of the labeled substrates were quantified to generate Figures 7B and 7C. TISSUE HOMOGENIZATION oleoyl-CoA DAG 5 minutes 10 minutes Scintillation counting TAG TAG Figure 7: Alternative mechanisms for triacylglycerol synthesis in the small intestine of Dgat1-/- mice. Since it was found that DGAT1 was not necessary for chylomicron synthesis, if the alternate pathways for TAG synthesis were assessed (DGAT2 expression, DGAT & DAG transacylase activity), then researchers would likely find results that support the theory that these mechanisms help compensate for the lack of DGAT1 in Dgat1-/- mice. A. Dgat2 expressed in all segments of small intestine with higher expression in proximal regions and lower expression in more distal regions. B. DGAT activity were 50% lower in more distal regions of the small intestine. DGAT activity in Dgat1-/- mice were 10-15% of levels in WT mice. C. Similar DAG transacylase activity in the small intestine of WT and Dgat1-/- mice.  Discussion 1. Does DGAT1 deficiency alter triacylglycerol metabolism in enterocytes? - DGAT1 was not essential for quantitative dietary triacylglycerol absorption. 2. Can mice that lack DGAT1 synthesize chylomicron-sized lipoprotein particles? - Dgat1-/- mice had reduced postabsorptive chylomicronemia and accumulated neutral-lipid droplets in the cytoplasm of enterocytes when chronically fed a high fat diet. 3. What are the relative contributions of DGAT1, DGAT2, and DAG transacylase in intestinal TAG synthesis and absorption? - If DGAT1 is absent, at least 2 enzymes: DGAT2 andDAG transacylase will help to catalyze TAG synthesis in mouse intestine. This compensate for the lack of DGAT1. - Highest activity of DGAT happens in the proximal intestine. DGAT1 is not important for intestinal TAG metabolism when dietary load of fat was high.  Conclusions In the absence of DGAT1, there is no dietary fat found in the large intestine, suggesting that DGAT1 is not necessary for the small intestine to absorb dietary triglycerides. TAG synthesis and absorption is an highly protected process, highlighted by the redundant and widespread mechanisms involved. This can likely be attributed to evolutionary pressure to maximize rich energy sources in times of food scarcity. There are other enzymes which are able to catalyze the synthesis of triglycerides, which accounts for the fact that fasted plasma TAG levels are similar in WT and Dgat1-/- mice. However, when dietary fat is high, these mechanisms cannot compensate at a fast enough rate, leading to decreased chylomicronemia and increased neutral lipid droplets in enterocytes.  Future Directions Based on the findings of this paper, more research is needed to determine the specific roles of DGAT2 and DAG transacylase in TAG synthesis and absorption. This paper showed that at least two additional enzymes compensated for the loss of DGAT1 function in Dgat1-/- mice, but the relative contribution of these enzymes remains unknown. Future experiments may be conducted on Dgat2-/- and DAG transacylase-/- mice in order to elucidate whether knocking out either of these enzymes impairs TAG resynthesis and chylomicronemia. Based on the evolutionary necessity of TAG absorption for survival, the researchers suggested that multiple redundant mechanisms for TAG processing may be present. Interesting relevant findings since this paper was published in 2002… “DGAT exists in two isoforms: DGAT-1 (expressed ubiquitously) and DGAT-2 (expressed primarily in the liver, intestine and white adipose tissue). While DGAT-2 deficiency is lethal [202,203], experimental DGAT-1-deficient phenotypes are viable and have intermediate (heterozygous) or complete (homozygous) resistance to diet-induced obesity, insulin resistance and hepatic steatosis [203–206]. Hence, DGAT-1 inhibition has emerged as a potential therapeutic approach for hypertriglyceridemia and insulin resistance.” (Sahebkar et. al, 2014) “[In] mice with global and liver-specific knockout of Dgat1…DGAT1 was required for hepatic steatosis induced by a high-fat diet and prolonged fasting, which are both characterized by delivery of exogenous fatty acids to the liver. Studies in primary hepatocytes showed that DGAT1 deficiency protected against hepatic steatosis by reducing synthesis and increasing the oxidation of fatty acids.” (Villanueva et. al, 2009) “Mice lacking DGAT1 are viable with reduced fat stores of TGs, whereas DGAT2 KO mice die postnatally just after birth with >90% reduction of TGs, suggesting that DGAT2 is the predominant enzyme for TG storage…DGAT1 and DGAT2 can largely compensate for each other for TG storage but that DGAT1 uniquely has an important role in protecting the ER from the lipotoxic effects of high-fat diets.” (Chitraju et. al, 2019) Thank you for listening! Questions?