Coenzyme A-independent Monoacylglycerol Acyltransferase from Rat Intestinal Mucosa

Abstract

Rat intestinal mucosa contains high diacylglycerolsynthesizing activity (monoacylglycerol acyltransferase (MGAT) activity) due to monoacylglycerol and fatty acid, independently of coenzyme A and ATP. MGAT activity was purified from rat intestinal mucosa by successive chromatography separations on DEAEcellulose, CM-Sephadex, and anti-IgG-Sepharose against rat pancreatic lipase. The enzyme was electrophoretically homogeneous, and its molecular weight was 49,000, which is identical with that of rat pancreatic lipase. Immunoblotting analysis with antibody against rat pancreatic lipase showed one immunoreactive protein with an estimated molecular weight of 49,000. The activity of the purified enzyme was completely inhibited by addition of the antibody. Using immunocytochemical techniques, it was found that immunoreactive protein against rat pancreatic lipase was uniformly distributed within the absorptive cells of the intestine but was absent from the microvillar membrane. The MGAT activity of intestinal mucosal homogenate was inhibited by about 65% by addition of antibody against rat pancreatic lipase. Trioleoylglycerol-and dioleoylglycerol-hydrolyzing activities of the purified enzyme and pancreatic lipase were inhibited by addition of intestinal mucosa extract.These results suggest that pancreatic lipase is present in intestinal absorptive cells and that it may contribute to resynthesis of diacylglycerol from monoacylglycerol and fatty acids in these cells.Dietary fat (triacylglycerol) is digested by pancreatic lipase to 2-monoacylglycerols and free fatty acids prior to its absorption in the intestinal lumen (1). Unlike esterases, pancreatic lipase develops its full activity only with emulsified substrates; i.e. it is activated at lipid-water interfaces (2). The adsorbed 2-monoacylglycerols and free fatty acids are resynthesized to triacylglycerol by enterocytes. Triacylglycerols are synthesized by two pathways, the glycerol 3-phosphate pathway and the monoacylglycerol pathway (3). During fat absorption in monogastric animals, the monoacylglycerol pathway is the major route of triacylglycerol synthesis (4). This pathway involves the stepwise acylation of 2-monoacylglycerols absorbed from the intestinal lumen. Monoacylglycerol acyltransferase (MGAT) 1 is a microsomal enzyme that catalyzes the synthesis of 1,2-diacylglycerols from 2-monoacylglycerols and long chain fatty acyl coenzyme A (5, 6). Fatty acyl coenzyme A is synthesized from free fatty acid and coenzyme A by fatty acyl-CoA synthetase, a reaction which requires energy (ATP). Because the main purpose of eating fat diets is to gain energy, it is comprehensible that ATP is required in the fat adsorption process. Therefore, we examined a newly discovered pathway which does not require energy.Previously, we reported that, under physiological conditions, lipases/esterases catalyze the synthesis of acyl-alcohol ester from free fatty acids and alcohols (7-9). These reactions do not require ATP or coenzyme A. Pancreatic cholesterol esterase, which is known to hyd...

Monoacylglycerol Metabolism in Human Intestinal Caco-2 Cells

“…Recently, however, another potential mechanism for intestinal TG formation was proposed. Tsujita et al suggested that PTL may facilitate a CoA-independent pathway of DG synthesis from MG and FFA in rat intestinal mucosa, by showing that a CoA-independent MGAT activity was inhibited up to 65% by addition of the antibodies against PTL (28). In the present studies, we were unable to detect a protein immunoreactive to polyclonal anti-human PTL antibodies either in Caco-2 cells or in rat jejunal mucosa homogenates.…”

“…Western Blot for Human PTL-Tsujita et al (28) and Mahan et al (29) have reported that PTL is expressed in the absorptive cells of the rat intestine. Because PTL may, therefore, represent an alternative mechanism whereby exogenous MG could be metabolized independent of the MGAT pathway, the expression of human PTL in Caco-2 was analyzed by Western blot to determine whether these cells express a protein immunoreactive to a polyclonal anti-human PTL antibody.…”

Carboxylesterase 3 (EC 3.1.1.1) Is a Major Adipocyte Lipase

“…Carboxylesterases are present in a wide variety of organs and tissues of mammals and are implicated in the pharmacokinetics of drugs containing ester or amide bonds (17). Carboxylesterase activity has been reported in rat WAT (18,19). To our knowledge, this is the first identification of esterase 1 in WAT.…”

Inhibitory effects of chondroitin sulfate prepared from salmon nasal cartilage on fat storage in mice fed a high-fat diet

“…Moreover, the present investigation was designed to clarify whether or not chondroitin sulfate prevented the obesity induced by longer-term feeding of a highfat diet (8 weeks). [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C] Palmitic acid (2.1 GBqammol) and [9, H] trioleoylglycerol (991.6 GBqammol) were obtained from Du Pont NEN. purchased from Sigma (St. Louis, MO).…”

“…The ®lters were then dissolved in ACS II according to the supplier's instructions (Amersham Japan, Tokyo, Japan) and their radioactivity was measured. For the palmitic acid absorption test, donor vesicles composed of phosphatidylcholineapalmitic acid (93 : 7, molar ratio) and a trace of [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C] palmitic acid were made by sonicating these substances in buffer A. The donor vesicles (29 nmol palmitic acid; 160,000 dpm) were incubated with the brush border membrane vesicles (48 mg of protein) for 30 min at 20 C, and then the donor vesicles were separated from the brush border membrane by ®ltration as described above.…”