(Received for publication, August 28, 1995; and in revised form, November 9, 1995)
From the
Rat intestinal mucosa contains high diacylglycerol-synthesizing 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 DEAE-cellulose, 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) ()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, 8, 9) . These reactions do not require ATP or coenzyme A. Pancreatic cholesterol esterase, which is known to hydrolyze cholesterol ester, catalyzes its resynthesis from cholesterol and free fatty acids within intestinal cells(10, 11) . In this paper, we report the purification and characterization of diacylglycerol synthesizing enzyme prepared from rat intestinal mucosa. We compare the molecular and catalytic properties of this enzyme and pancreatic lipase prepared from rat pancreas. It is concluded that the two enzymes are either identical or very similar. From these results, we demonstrate that pancreatic lipase digests dietary fats to 2-monoacylglycerols and free fatty acids in the intestinal lumen and then resynthesizes diacylglycerols from these compounds in the intestinal adsorption cells.
The supernatant was applied to a column of
DEAE-cellulose (1.9 23 cm) equilibrated with buffer 1. Most
enzyme activity was recovered in the nonadsorbed fraction. The active
fractions were pooled, dialyzed against 40 mM potassium
phosphate buffer, pH 6.1, and applied to a CM-Sephadex C-50 column (1.9
20 cm) equilibrated with the same buffer. The protein was
eluted from the column with a linear gradient of phosphate buffer (40
to 400 mM), pH 6.1. The active fractions were pooled, dialyzed
against 150 mM potassium phosphate buffer, pH 7.2, containing
100 mM NaCl, and loaded on to a column of anti-pancreatic
lipase IgG equilibrated with the same buffer. The protein and the gel
were incubated together overnight at 4 °C under gentle agitation.
Then, the column was washed successively with equilibrated phosphate
buffer, Triton X-100 buffer (150 mM phosphate buffer, 100
mM NaCl, and 0.2% Triton X-100, pH 7.2) and then again with
the original buffer. The active protein was eluted with a 200 mM glycine-HCl buffer, pH 2.8. Each fraction was immediately
neutralized with 0.1 N NaOH. The active fractions were pooled,
dialyzed against 10 mM potassium phosphate buffer, pH 7.0, and
stored at -80 °C.
In studies on the
effect of fatty acid chain length, the activity was also determined
using [C]monooleoylglycerol. Incubations were
carried out at pH 7.0 for 30 min, and diacylglycerols were extracted by
the method described above. The upper heptane phase (1 ml) was dried
with a stream of nitrogen, solubilized in 50 µl of hexane, and
separated by thin layer chromatography (Whatman K-5 silica gel plates)
with a hexane:diethyl ether:acetic acid (60:40:1, v/v). Lipids were
located with iodine vapor, and spots of diacylglycerol were scraped off
for measurement of their radioactivity.
Trioleoylglycerol- and
dioleoylglycerol-hydrolyzing activities were determined by measuring
the rate of release of [H]oleic acid. The assay
mixture consisted of 0.2 ml of 100 mM potassium phosphate
buffer, pH 7.0, containing 0.3 µmol of
[
H]trioleoylglycerol (120,000 dpm) or 0.3
µmol of [
H]dioleoylglycerol (80,000 dpm) and
1 mg of gum arabic. Incubations were carried out at 37 °C for 30
min, and the free oleic acid was extracted and determined by the method
of Belfrage and Vaughan (16) .
MGAT activity was determined in the absence of coenzyme A and
ATP. The tissue distribution of MGAT activity in the rat was measured
by homogenizing and centrifuging the tissue at 1,000 g and assaying the supernatant (Table 1). The highest MGAT
activity was observed in the intestinal mucosa, 34-120-fold that
found in other tissues. The MGAT activity of intestinal mucosa extract
was proportional to the amount of protein and the incubation time (data
not shown). The enzyme concentration dependence was not changed by
addition of coenzyme A and ATP (data not shown). Over a 15-min
incubation period, the activity decreased slightly with time when
coenzyme A and ATP were added.
Rat intestinal mucosa (78 g) were homogenized, extracted, fractionated by ammonium sulfate, and applied to a DEAE-cellulose column. The enzyme did not adsorb to the column, and the active wash fraction was pooled and applied to a CM-Sephadex C-50 column (Fig. 1A). The MGAT activity was completely bound and eluted a single peak, developed with a linearly increasing gradient of phosphate concentration. Active fractions (tubes 11-17) were pooled, dialyzed, and applied to an anti-IgG Sepharose column. The enzyme was completely bound and eluted with glycine buffer, pH 2.8 (Fig. 1B).
Figure 1:
A, ion exchange chromatography of rat
intestinal MGAT on a CM-Sephadex C-50 column. The nonadsorbed enzyme
fractions from DEAE-cellulose were applied to the column. The column
was washed with buffer and developed with a linear gradient of
phosphate buffer (40 to 400 mM) (arrow). B,
immunoaffinity chromatography of rat intestinal MGAT on
anti-IgG-Sepharose gel. The active proteins from the CM-Sephadex C-50
column were incubated with anti-IgG-Sepharose against rat pancreatic
lipase. The gel was washed with phosphate buffer, Triton X-100 buffer,
and then again with the phosphate buffer. The active proteins were
eluted with 200 mM glycine-HCl buffer, pH 2.8 (arrow). A (
) and MGAT activity
(
) were measured.
The enzyme purification steps and the enzyme yield at each step are summarized in Table 2. At the final step of purification, the specific MGAT activities was 3,210 nmol/mg of protein/min. Purified enzyme hydrolyzed trioleoylglycerol and dioleoylglycerol, whose specific activities for trioleoylglycerol and dioleoylglycerol were 35,900 and 43,500 nmol/mg of protein/min, respectively. The purified enzyme gave a single band on SDS-polyacrylamide gel electrophoresis from which its molecular weight was estimated to be 49,000 (Fig. 2). Pancreatic lipase purified from rat pancreas also gave a single band, having the same molecular weight. Immunoblotting analysis with antibody against rat pancreatic lipase showed one immunoreactive protein with an estimated molecular weight of 49,000 (Fig. 2). The purified enzyme had an isoelectric point of pH 6.8, which was identical with that found for rat pancreatic lipase (data not shown).
Figure 2: SDS-polyacrylamide gel electrophoresis of MGAT from rat intestinal mucosa protein obtained from CM-Sephadex (approximately 37 µg) (lanes 1 and 4) and anti-IgG Sepharose (approximately 3.8 µg) (lanes 2 and 5) columns and purified rat pancreatic lipase (approximately 17 µg) (lane 3). The gel was stained with Coomassie Brilliant Blue R-250 (lanes 1, 2, and 3) and analyzed by immunoblotting with anti-IgG against rat pancreatic lipase (lanes 4 and 5).
Purified enzyme was mixed with various amounts of anti-IgG prepared with rat pancreatic lipase, left to stand overnight at 4 °C, centrifuged to remove insoluble material, and the residual enzyme activities in the supernatant measured. MGAT activity in the supernatant decreased as the amount of anti-IgG increased (Fig. 3). Similar profiles were observed for the inhibition of trioleoylglycerol- and dioleoylglycerol-hydrolyzing activities (data not shown). All three activities were unaffected by nonimmune IgG, which was used as a control (data not shown).
Figure 3: Effect of the rat pancreatic lipase antibody on the MGAT activity of purified rat intestinal MGAT. The purified MGAT (1.2 µg) was mixed with various amounts of anti-IgG against rat pancreatic lipase. After overnight incubation at 4 °C, the mixture was centrifuged to remove insoluble material. Residual MGAT activity was then measured.
The incorporation of 2-monooleoylglycerol and oleic acid into dioleoylglycerol were determined. With a constant 2-monooleoylglycerol level of 0.5 mM, dioleoylglycerol formation increased with the concentration of oleic acid (Fig. 4A). With oleic acid constant at 0.4 mM, the enzyme activity increased with the concentration of 2-monooleoylglycerol up to 0.5 mM and then decreased (Fig. 4B). Table 3shows the effect of fatty acid chain length on diacylglycerol synthesis by the purified enzyme. With the saturated fatty acid tested, its activity was maximal with myristic acid and fell with decreasing fatty acid chain length. The MGAT activity for caproic acid was not detected. The activity for oleic acid was 8-fold that for stearic acid. The enzyme activity for 2-monoacylglycerol was about 3 to 6 times that for 1-monoacylglycerol (Table 4). With ether analogs of monoacylglycerol as substrates, the MGAT activity for glycerol ether substituted at position 2 was essentially identical with that for glycerol ether substituted at position 1.
Figure 4: Effect of substrate concentration on MGAT activities. Purified rat intestinal MGAT was incubated with various amounts of oleic acid and 2-monooleoylglycerol. A and B show plots of reaction rate versus substrate concentration with substrate of oleic acid and monooleoylglycerol, respectively. In A, the monooleoylglycerol concentration was 0.5 mM, and, in B, the oleic acid concentration was 0.4 mM.
Photomicrography revealed a brown reaction on the anti-pancreatic lipase IgG-treated sections of small intestine. The reaction product was consistently visualized within adsorptive cells (Fig. 5A). The submucosa of these sections were also stained strongly by 3,3-diaminobenzidine tetrahydrochloride (data not shown). On adjacent sections treated with nonimmune IgG, little or no reaction was observed with absorptive cells (Fig. 5B) or other components (data not shown).
Figure 5:
Photomicrograph of immunohistochemically
stained section prepared using anti-pancreatic lipase IgG in intestinal
absorptive cells of rats treated with antibody against rat pancreatic
lipase (A) and control IgG (B). Magnification,
780.
Anti-IgG against rat pancreatic lipase blocked the MGAT activity of rat intestinal mucosa extract in a dose-dependent manner; about 65% activity was inhibited by addition of the antibody (data not shown). Control nonimmune IgG did not affect MGAT activity in the intestinal mucosa (data not shown). Trioleoylglycerol-hydrolyzing activity of the purified enzyme and pancreatic lipase was inhibited in a dose-dependent fashion by addition of intestinal mucosa extract (data not shown). Dioleoylglycerol-hydrolyzing activity was also inhibited by the addition of intestinal mucosa extract (data not shown).
In this paper, we purified MGAT from rat intestinal mucosa and found that its activity did not depend on coenzyme A and ATP. The purified enzyme might be identical with rat pancreatic lipase, as concluded from the following observations: (a) the purified enzyme had MGAT activity and triacylglycerol- and diacylglycerol-hydrolyzing activities; (b) antibody against rat pancreatic lipase inhibited the activity of MGAT prepared from rat intestinal mucosa (Fig. 3); (c) the band of purified enzyme on SDS-polyacrylamide gel electrophoresis was identical with that of rat pancreatic lipase, and it was detected by immunoblotting analysis using antibody against rat pancreatic lipase (Fig. 2); (d) immunoreactive protein against rat pancreatic lipase was distributed within intestinal cells (Fig. 5).
We have
previously reported that lipase(s) and esterase(s) synthesize fatty
acyl esters. Carboxylesterase from rat adipose tissue can synthesize
fatty acid ethyl esters from free fatty acid and ethanol (7) .
Lipoprotein lipase and carboxyl ester lipase also synthesize their
esters from fatty acid and ethanol(8, 9) . The
mechanism of fatty acid ethyl ester formation by the enzyme is as
follows. When the enzyme was incubated with fatty acid and ethanol, an
acyl-enzyme intermediate was formed and fatty acid ethyl ester was
synthesized though nucleophilic displacement by ethanol. Exchange of
fatty acid carboxyl oxygens with water oxygens was demonstrated by
Muderhwa et al.(18) using O water. They
suggested that fatty acids are good ``substrates'' for
lipase(s). Mechanistic studies indicated that reactions catalyzed by
the enzyme proceed via an acyl-enzyme intermediate. The primary
structure of these enzymes contains Gly-Xaa-Ser-Xaa-Gly, which is the
common active site sequence of a serine enzyme(19) . The
catalytic mechanism of these enzymes resembles that of serine
proteinase catalysis, which proceeds via an acyl-enzyme intermediate.
Pancreatic lipase is also a serine enzyme(20) . It catalyzes
the synthesis of fatty acid ethyl ester from fatty acid and
ethanol(21) . In this paper, we demonstrated that pancreatic
lipase catalyzed the synthesis of diacylglycerol from free fatty acid
and 2-monoacylglycerol. In this reaction, it was supposed that
diacylglycerol was also synthesized through nucleophilic displacement
by monoacylglycerol via an acyl-enzyme intermediate.
The lipase(s) is reported to synthesize some esters in organic or alcohol media. Porcine pancreatic lipase catalyzes transesterification between tributyl glycerol and various primary and secondary alcohols in a 99% organic medium(22) . Microbial lipase(s) also synthesize oleoylglycerols from oleic acid and glycerol in an 80% glycerol medium (23) . Proteases also catalyze the synthesis of peptide bonds in organic media(24) . These serine enzymes are used industrially for ester or peptide synthesis in organic media. In an organic medium, which contains a very low concentration of water, the enzyme also forms an acyl-enzyme intermediate, and the esters are easily synthesized by nucleophilic displacement by alcohols instead of water. In other word, ``alcoholysis'' rather than ``hydrolysis'' of the acyl-enzyme intermediate took place. Usually, the reaction site of lipase is the lipid-water interface, such as the plasma membrane or lipoprotein surface in an aqueous medium. Alcohols such as ethanol or monoacylglycerol might more easily approach the lipid-water interface than water because alcohols are more hydrophobic than water. Therefore, lipase(s) might be able to synthesize the esters in an aqueous medium at the lipid-water interface. In this study, we demonstrated that pancreatic lipase catalyzes diacylglycerol synthesis in an aqueous medium.
Dietary fat (triacylglycerol) is hydrolyzed by pancreatic lipase to 2-monoacylglycerols and free fatty acids prior to its absorption by the intestinal villus cells. In enterocytes, these products enter the monoacylglycerol pathway, the predominant route for acylglycerol biosynthesis in the absorptive cells. The sequential intracellular re-esterification of 2-monoacylglycerol to triacylglycerol is catalyzed by a triacylglycerol synthetase complex(25, 26) , which distributes intestinal microsomes and consists of MGAT, diacylglycerol acyltransferase, and acyl-coenzyme A synthetase. MGAT (monoglyceride acyltransferase, Enzyme Nomenclature EC 2.3.1.22) catalyzes the synthesis of 1,2-diacylglycerols from 2-monoacylglycerols and long chain fatty acyl-coenzymes A. High levels of activity were observed in intestinal mucosa and liver microsomes of suckling rats(4, 5) . In the present study, we demonstrated that pancreatic lipase might be distributed within intestinal cells and that it had MGAT activity independently of coenzyme A and ATP. The specific MGAT activity (3.21 µmol/mg of protein/min) of pancreatic lipase was about three times that of purified MGAT from rat liver (1.22 µmol/mg of protein/min) (27) . The presence of ester-synthesizing activity as a reverse catalytic activity of lipase is well known. The specific activity of the forward reaction (diacylglycerol hydrolysis, 43.5 µmol/mg of protein/min) was about 14 times that of the reverse reaction (MGAT activity). However, the diacylglycerol-hydrolyzing activity of the lipase was inhibited by addition of intestinal mucosa extract. Indeed, when free fatty acid and 2-monooleoylglycerol were incubated with rat intestinal mucosa extract, diacylglycerols were formed to an extent depending on dose and time. These activities were not activated by the addition of ATP and coenzyme A. Furthermore, these activities were inhibited in intestinal mucosa extract by addition of the antibody against rat pancreatic lipase.
Bosner et al.(28) demonstrated that human pancreatic lipase bound to intestinal cell membranes and modulated the uptake of fatty acid derived from triacylglycerols cleaved by the lipase. They also suggested that the binding site was composed of a heparin which is a component of the brush-border membrane. However, MGAT activity in the intestinal mucosa was not changed by washing it with heparin (100 units/ml) or high concentrations of NaCl (2 M) (data not shown). Furthermore, in an immunocytochemical study using antibody against rat pancreatic lipase, immunoreactive product was not observed on the microvillar surface (Fig. 5), although it was observed within absorptive cells. Similar distribution was observed for another pancreatic enzyme, cholesterol esterase. Gallo et al.(29, 30) demonstrated that ``intestinal'' cholesterol esterase was of pancreatic origin and was localized within the absorptive cells. They proposed two roles for pancreatic cholesterol esterase in the process of adsorption of dietary cholesterol in the intestine; catalysis of the hydrolysis of dietary cholesterol ester and maybe the catalysis of cholesterol ester re-esterification after being taken up by the villus cell. In this study, we propose two roles for pancreatic lipase in the process of dietary fat adsorption, like a cholesterol esterase; the digestion of dietary fats to 2-monoacylglycerols and fatty acids in the intestinal lumen, and possibly the catalysis of diacylglycerol resynthesis from 2-monoacylglycerol and free fatty acid in the intestinal absorptive cells.