From the Department of Biology (Area 3), University of York, York YO10 5YW, United Kingdom
Received for publication, February 11, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The Ka and Jmax of the facilitated component are 2- and 3-fold, respectively, of those for SGLT1 in vivo. After a meal, the average concentration across the luminal contents of rat jejunum reaches 48 mM (4), close to the Km of the facilitated component, and much higher concentrations may well be present locally at the brush-border membrane, as a result of the hydrolysis of complex dietary sugars (5). The facilitated component therefore appears to provide the major route by which glucose is absorbed immediately after a meal. Moreover, the activation of GLUT2 and its rapid trafficking to and from the brush-border membrane in response to changing glucose concentrations provides a cooperative mechanism by which absorptive capacity is matched precisely to dietary intake.
GLUT2 transports not only glucose but also fructose
(6). Fructose absorption across
the brush-border membrane is therefore mediated by both GLUT5, which is
specific for fructose, and GLUT2
(7). Phloretin, which inhibits
GLUT2 but not GLUT5, may be used separate out the contributions of each
transporter to fructose transport. In this way, we were able to show that a
4-fold stimulation of fructose absorption by PMA in isolated loops in
vitro was matched by a 4-fold increase in the level of GLUT2 at the
brush-border membrane and correlated with the activation of PKC II
(7). The level of GLUT5 and its
contribution to absorption did not change significantly.
Support for the model of rapid, GLUT2-mediated up-regulation of sugar absorption has been provided by the observation that glucagon-like peptide 2 promotes rapid insertion of GLUT2 into the brush-border membrane (8). Moreover, a definitive confirmation of the model has recently been provided by the observation that when the intestine of wild type mice is challenged with sugars by gastric intubation, a large increase in fructose transport occurs within minutes; the increase in transport does not occur in GLUT2-null mice and is attributable entirely to GLUT2 (9).
The intrinsic activity of PKC II is controlled by phosphorylation at
three sites, one in the activation loop and two in the C-terminal region,
located at Thr-500, Thr-641, and the hydrophobic site Ser-660, respectively
(1012).
Phosphorylation at all three sites serves to lock the enzyme into a
catalytically competent conformation, which is then regulated by
membrane-bound phosphatidylserine and also by second messengers
Ca2+ and diacylglycerol. The first step in the
production of mature PKC
II is phosphorylation of Thr-500 by PDK-1,
which is blocked by inhibitors of PI3-kinase, since the latter regulates PDK-1
(1315).
Phosphorylation of Thr-500 then permits autophosphorylation of Thr-641 and
Ser-660 (16). Activation of
PKC
II is tightly coupled to phosphorylation of the hydrophobic site
Ser-660, which greatly increases affinity for Ca2+
(17). Phosphorylation of the
hydrophobic site in PKC
and PKC
is sensitive to rapamycin
(18,
19), suggesting a role for
mTOR in controlling phosphorylation at this site, possibly by regulating the
activity of a phosphatase
(12).
In our previous work, we implicated PKC II in the PMA-induced
stimulation of the GLUT2-mediated component of fructose absorption by using
chelerythrine, which inhibits PKC
II activity by binding directly to the
catalytic site (7). In the
present work, we have now used inhibitors of PI3-kinase and mTOR, wortmannin
and rapamycin, respectively, to inhibit the activities of the intracellular
signaling pathways that stimulate the phosphorylation and intrinsic activity
of PKC
II. Using this quite different approach, we have reproduced and
extended our previous results to confirm the involvement of PKC
II in
the regulation of GLUT2 trafficking to the brush-border membrane. The data
suggest new mechanisms for the regulation of fructose and glucose absorption
by insulin and amino acids.
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EXPERIMENTAL PROCEDURES |
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Perfusion of Jejunal LoopsRats were anesthetized by an
intraperitoneal injection of a mixture of 1.0 ml of Hypnorm (Janssen) and 0.4
ml of Hypnovel (Roche Applied Science) per kg of body weight. The systems for
the luminal perfusion of jejunal loops in vitro and in vivo
have been described in detail previously
(2,
7,
20). Because PKC activators
and inhibitors take some time to become effective in whole tissue and because
there is a limit to the time of viability for isolated loops in
vitro, the following procedure for the treatment of jejunum with drugs
was adopted. Jejunum was first perfused luminally in vivo with 5
mM D-fructose (plus 1 mM
-hydroxybutyrate as an energy source) in the presence or absence of
drugs; the perfusion system was a gas-segmented, single pass system with
perfusate and gas flow rates of 0.75 and 0.38 ml
min1, respectively. For each and every perfusion,
fructose was present in the perfusate throughout the entire perfusion. Jejunum
was perfused with 200 nM PMA for 30 min or with chelerythrine for
45 min; when they were perfused in combination, PMA was added to the perfusate
15 min after the inhibitors. At the end of the in vivo treatment
period, the cannulated loop was then excised and perfused in vitro
using the gas-segmented, recirculated flow system: 5 mM
D-fructose and the combination of drugs at the end of in
vivo treatment period were perfused luminally through the jejunum for 60
min; the flow rates of perfusate and gas were 7.5 and 3.4 ml
min1, respectively. Control perfusions were
performed in which no drugs were present; these showed that there was no
falling off in the D-fructose uptake rate after the initial
approach to the steady-state, confirming that this approach to drug treatment
and perfusion ensured that the jejunum was viable for the whole experimental
period from the start of the in vivo perfusion to the finish of the
in vitro perfusion. Samples (0.05 ml) were taken from the perfusate
at 5-min intervals throughout the in vitro perfusion. After
measurement of fructose concentration with a COBAS automatic analyzer using a
test kit (Roche Applied Science), the amount of fructose remaining in the
perfusate was calculated with correction for losses in perfusate volume caused
by water transport. Because the perfusate is recirculated, the concentration
of fructose decreases with time; it was therefore necessary to limit the
period over which the rate of transport was measured to one in which the
concentration decreased by no more than 20%. Over such periods, plots of
luminal perfusate content versus time are effectively linear. The
rate of fructose transport, expressed as µmol
min1 (g of dry
weight)1, was therefore determined by linear
regression analysis as the average rate of disappearance over this period from
the luminal perfusate in vitro.
Membrane Vesicle PreparationBrush-border membrane vesicles
were made as described previously
(20); every stage of the
preparation was performed at 04 °C to prevent changes in
trafficking after the intestine had been excised. Briefly, the jejuna of two
rats were perfused with drugs as described above. The timing of the final
in vitro perfusion was such as to get as direct a correspondence as
possible between rates of transport and extent of trafficking; perfusions for
vesicle preparations were therefore terminated at a time point corresponding
to half the time period over which the average rate of transport was
determined as described above. Immediately after perfusion, each jejunum was
flushed with ice-cold buffered mannitol (20 mM imidazole buffer, pH
7.5, containing 250 mM mannitol and 0.1 mM
phenylmethylsulfonyl fluoride) to arrest trafficking of GLUT2 and to trap any
labile intermediates of PKC II at the membrane. Jejunum was then placed
on an ice-cold glass plate and slit longitudinally so that the muscle of the
jejunum flattened out on to the cold plate. Mucosal scrapings were taken with
an ice-cold glass slide and homogenized immediately at 4 °C in buffered
mannitol using a Kinematica Polytron homogenizer (4x 30-s bursts using
the large probe at setting 7). The rest of the preparation and its detailed
characterization for purity are as described by Corpe et al.
(20). At the end, the
preparation was revesiculated and rinsed three times to ensure that all
cytosolic contaminants were removed. Enrichment of sucrase activity in these
highly purified preparations ranged from 16- to 20-fold; there was no
significant enrichment of Na+/K+-ATPase activity.
Dephosphorylation of brush-border membrane vesicles with protein phosphatase
1A (PP1A) was performed as described by Keranen et al.
(10).
Affinity Purification of PKC IIAll
procedures took place at 4 °C. C10 antibody was coupled to protein
A-Sepharose 4B fast flow resin (Sigma) with disuccinimidyl suberate and packed
into a Bio-Rad Econocolumn (0.5 x 0.5 cm). The column was washed and
stored in phosphate buffered saline (phosphate-buffered saline, pH 7.4) until
use. Brush-border membrane vesicles were solubilized to 0.5 mg of
protein/ml1 in lysis buffer (150 mM
NaCl, 25 mM KCl, 1 mM EDTA, 1 mM EGTA, 1
mM Na2VO3,25mM NaF, 0.1
mM phenylmethylsulfonyl fluoride, and 25 mM HEPES, pH
7.2, plus 1% (w/v) Triton X-100, 1% (w/v) deoxycholate, and 0.2% (w/v) SDS and
centrifuged at 13,000 rpm to separate soluble and insoluble fractions. The
soluble fraction was diluted 1:1 with phosphate-buffered saline (pH 7.4) and
precleared on an antibody-free protein A column. The flow-through (protein
eluate) from this column was loaded onto the antibody column at <0.2 ml
min1, after which the column was washed (150
mM NaCl, 25 mM Tris-HCl, pH 7.2) to remove unbound
proteins. Bound proteins were then eluted with 1 M NaCl, 25
mM Tris-HCl (pH 7.2), plus 0.2% (w/v) SDS. The collected fractions
were precipitated with 6% (v/v) perchloric acid and then spun down, and the
pellet was resuspended in SDS sample buffer. Separated samples were
transferred to polyvinylidene difluoride membrane by Western blotting and
immunoblotted using a polyclonal antibody to ubiquitin raised in rabbit kindly
supplied by Professor R. J. Mayer (Nottingham).
Western BlottingSDS-PAGE and Western blotting was performed
as described previously using ECL (enhanced chemiluminescence) detection
(7,
20). C10 antibody to the last
10 C-terminal amino acids of PKC II
(Fig. 1) was raised in rabbit
by Professor N. Groome (Oxford Brookes University). A C18 antibody to the last
18 amino acids of PKC
II (Fig.
1, not phosphorylated at Ser-660) was purchased from Santa Cruz
Biotechnology, Santa Cruz, CA. Antibodies to the last 14 amino acids at the C
terminus of GLUT2 and GLUT5 were raised in rabbits. All antibodies were
polyclonal. Neutralization of each antibody by preincubation with excess of
the corresponding antigenic peptide abolished labeling, confirming the
specificity of the antibodies. Quantitation of Western blots was performed
using a Flowgen AlphaImager 1200 analysis system (Alpha Innotech Corp.).
Protein levels (as indicated by band intensities) determined in vesicle
preparations from jejunum were expressed relative to those in control
preparations. A liver GLUT2 standard was routinely used in blotting
experiments. The linear range of intensity response in ECL photographs was
established using a 20-fold range of the amount of an actin standard
(240 µg). After background correction, the response was linear
(correlation coefficient 0.996) for integrated density values ranging from
13,024 to 465,029. As much as possible, exposures were such that the intensity
values fell within the middle third of the linear response range. The same
loading of 15 µg of protein was used for all samples. Comparison of
relative levels of target proteins was made on a total protein basis to
minimize potential complications that might be caused by the trafficking of
other proteins in response to the same stimuli that affect the trafficking of
target proteins.
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Statistical AnalysisValues are presented as means + S.E. and were tested for significance using Student's t test.
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RESULTS |
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In that previous work with chelerythrine, we provided evidence that the
increase in GLUT2-mediated absorption induced by PMA is matched by a
corresponding increase of GLUT2 in the brush-border membrane. These changes
correlated with activation of PKC II, and there was little change in
GLUT5-mediated transport and GLUT5 level
(7). Vesicles were therefore
prepared from jejunum perfused first in vivo and in vitro to
determine the levels of transporters and PKC
II in response to rapamycin
and wortmannin. Protein trafficking was arrested by flushing with ice-cold
perfusate immediately before excision of jejunum
(7,
21). The timing of the final
in vitro perfusion was such as to get as direct a correspondence as
possible between rates of transport and extent of trafficking. Perfusions for
vesicle preparations were therefore terminated at a time point corresponding
to half the time period over which the average rate of absorption was
determined as described above. Vesicles were then Western blotted using
antisera to the C-terminal 14 amino acids of GLUT5 or GLUT2. GLUT5 appeared as
a triplet from 53 to 58 kDa and GLUT2 as a tightly spaced doublet from 61 to
63 kDa (Fig. 2). The distinct
banding patterns confirmed that there was no cross-reactivity between antibody
to one transporter and the other transporter; in addition, antibody to a
sequence within the extracellular loop of GLUT2 gave similar results to the
C-terminal antibody (8). All
bands were eliminated by preincubation of antiserum with excess of the
corresponding antigenic peptide, confirming the specificity of GLUT5 and the
GLUT2 antisera for their target transporters (data not shown).
Fig. 2 shows that PMA increased
the level of GLUT2 3.9 ± 0.6-fold as compared with control. Rapamycin
blocked PMA-induced trafficking of GLUT2 to the brush-border membrane so that
GLUT2 remained at control levels. GLUT5 levels were not significantly
different from control levels in any other condition. Wortmannin behaved
similarly to rapamycin.
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Western blots were performed with C18 antibody raised against the
C-terminal sequence residues 656673 of PKC II. C18 antibody
recognized a doublet at 80 and 78 kDa and a single band at 49 kDa. All bands
were eliminated by preincubation of antiserum with excess C18 peptide (data
not shown). PMA strongly increases the intensity of the 49-kDa band. It also
increases the intensities of the doublet, although to a lesser extent.
Translocation of conventional PKC isoenzymes to a target membrane is widely
taken as an indicator of activation, which is often accompanied by
calpain-dependent cleavage in the hinge region to a constitutively active
catalytic domain of
50 kDa. The activity of this form is independent of
any activators, such as diacylglycerol, phosphatidylserine, and
Ca2+ because of the separation of the catalytic unit
from the regulatory domain and coincidentally from the pseudosubstrate in the
N-terminal sequence (22,
23). Since the 80-/78-kDa
bands correspond to those for two control standards (rat brain homogenate and
a postnuclear liver membrane preparation, data not shown) and the standards
either do not show or show only a light 49-kDa band, the 80-/78-kDa species
must represent native (uncleaved) forms of the enzyme, and the 49-kDa band
must represent the C-terminal catalytic domain. PKC
II in intestine
appears to be particularly susceptible to cleavage, a phenomenon we have seen
under many other conditions. The catalytic domain does not possess binding
sites for diacylglycerol and phosphatidylserine in the membrane, yet is found
in vesicles. It should therefore be noted that at the end of a preparation,
vesicles are revesiculated several times through a 21-gauge needle so that the
presence of the catalytic domain in vesicles is not caused by simple, physical
trapping of cytosolic proteins as vesicles reseal during preparation. That we
detect the catalytic domain strongly in vesicles most likely reflects the fact
that jejunum is flushed with ice-cold perfusate immediately before excision to
prevent the rapid trafficking of GLUT2 away from the membrane. Coincidentally,
this procedure traps the catalytic domain cleavage product in association with
its (unknown) substrate. Rapamycin completely blocks the PMA-induced increase
in the intensity of the 49-kDa species and, on its own, decreases band
intensity as compared with control. Wortmannin decreased the PMA-induced
increase in the intensity of the 49-kDa species, although not as effectively
as rapamycin. When perfused alone, wortmannin had no effect on band intensity
as compared with control. PKC
II activation, as indicated by the
intensity of the 49-kDa band, correlates with the increase in brush border
GLUT2 level, as reported previously
(2,
3,
7).
Western blots of brush-border membrane vesicles from jejunum perfused first
in vivo and in vitro were also performed using C10
polyclonal antibody raised against the C-terminal sequence residues
664673 of PKC II. The banding pattern with C10 antibody was very
different from that with C18 antibody. The C10 antibody detects the 80-/78-kDa
doublet only very weakly. However, it recognizes the catalytic domain at
49-kDa strongly; in addition, it also recognizes strong bands at 42 and 180
kDa and a lighter band at 250+ kDa. All bands were eliminated by preincubation
of antibody with excess C10 peptide, confirming antibody specificity. Again,
PMA causes strong activation of PKC
II, as indicated by the marked
increase in the intensity of the 49-kDa band as compared with control; the
increase was paralleled by a concomitant decrease in intensity of the 42-kDa
band. Rapamycin completely blocked the effects of PMA so that the banding
pattern remained similar to the control. Rapamycin alone strongly diminished
the intensities of the 49- and 250-kDa bands, and all but eliminated the 42-
and 250+-kDa bands. Wortmannin exerts similar effects on the levels of GLUT2
and on the activation of PKC
II to those of rapamycin
(Fig. 2B). Again the
reciprocal relationship of the 49- and 42-kDa bands is clearly seen. The major
difference is that the effects of wortmannin alone are not as marked as those
of rapamycin. Thus, at least at the concentration of wortmannin used here,
there are clear 42-, 180-, and 250+-kDa bands.
All the bands detected by C10 and C18 antibodies represent different
species of PKC II. Thus, no bands are detected when antibody is first
preabsorbed with the corresponding antigenic peptide. BLAST searches of the
NCBI and SWISS-PROT databases with either the C18 or the C10 sequence reveal
only PKC
II. All the bands are related to each other as shown by the
fact that rapamycin alone significantly diminishes or eliminates them. Since
rapamycin and wortmannin both inhibit the phosphorylation of PKC, the
reciprocal relationship of the intensities of the 49- and 42-kDa bands
suggests the 42-kDa species is a dephosphorylated form of 49-kDa species.
Treatment of vesicles with PP1, which dephosphorylates all three C-terminal
phosphorylation sites, converts the 49-kDa species to the 42-kDa species and
implies that the 42-kDa species is likely to be fully dephosphorylated
(Fig. 3). That activation at
the membrane results ultimately in dephosphorylation is in keeping with the
fact that activation induces an open conformation, which increases the
sensitivity of the C terminus to dephosphorylation by more than 2 orders of
magnitude (24). In addition,
the isolated catalytic domain naturally has an open conformation
(14,
25).
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PKC species with apparent molecular weights much higher than that of the
native, uncleaved enzyme have been reported to be polyubiquitylated
intermediates on a pathway targeted for proteasomal degradation
(26,
27). The Western blot in
Fig. 4 (lane 1) shows
that brush-border membrane vesicles from PMA-perfused intestine contain
several ubiquitylated proteins, presenting for the most part as a broad smear
with occasional discrete bands. An affinity column, in which C10 antibody was
covalently linked to the support, was therefore used to purify the 180-kDa
species to show that this species of PKC II was strongly cross-reactive
with anti-ubiquitin (lane 2). In some blots, very faint bands could
also be seen at 250+ kDa.
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The data thus far are consistent with the existence of two processes: the initial one is calpain-dependent cleavage of the native enzyme at the membrane on activation, and the second one is a turnover and degradation pathway, which involves dephosphorylation of the 49-kDa species to the 42-kDa species. Sugars also activate the degradation pathway and interconversion of the different species. This was clearly demonstrated when jejunum was perfused solely in vivo with Krebs-Henseleit buffer alone (no sugar, Fig. 5); blots with C10 antibody showed a strong 49-kDa band and blots with C18 antibody showed the 80-/78-kDa doublet in addition to strong 49-kDa band. However, when jejunum was perfused with different sugar substrates, namely glucose (100 mM), mannitol (100 mM), fructose (100 mM), and maltose (200 mM), C10 antibody also detected a well defined 42-kDa band and an intense 180-kDa band, as well as a lighter 250+-kDa band. Thus, in the absence of luminal sugar in vivo (no in vitro perfusion), there is substantial cleavage of the native enzyme but effectively no turnover to species on the ubiquitin-dependent degradation pathway. However, luminal perfusion with sugars in vivo (no in vitro perfusion) results in the activation of the degradation pathway in minutes. Since the 42-kDa species is derived by dephosphorylation of the 49-kDa species (Fig. 3), it seems that proteasomal degradation follows dephosphorylation in agreement with Hansra et al. (24). The order of production of detected species from the 80-/78-kDa native enzyme therefore appears to be 49, 42, 180, and 250+ kDa. Our data, of course, cannot preclude the possibility that some polyubiquitylated degradation intermediates can be directly derived from the native enzyme, although it seems that proteolytic cleavage is favored in intestine.
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The turnover provoked by mannitol was surprising since it is not thought to
be transported. However, these effects were not caused by an increase in
osmolarity at the high sugar concentrations used as compared with buffer alone
since perfusion with 5 mM fructose in vivo was sufficient
to induce equally definite turnover; moreover, analysis of mannitol revealed
no detectable reducing sugars (data not shown). These observations therefore
point toward a possible sensing mechanism (see below). As reported previously,
however, the activation of PKC II and GLUT2 insertion into the
brush-border membrane was favored by glucose and fructose as compared with
mannitol.
Western blots reveal that both PI3-kinase and PDK-1, which phosphorylates
Thr-500 in the activation loop of PKC II, are present at the
brush-border membrane (Fig. 6).
Also present is PKB, which is also a substrate for PDK-1. The levels of all
three signaling enzymes in the presence of PMA are increased between 48 and
64% as compared with the corresponding control; levels in all other conditions
are similar to control. Thus, the necessary signaling enzymes for wortmannin
and rapamycin action are present. Interestingly, the upstream pathway is
activated by PMA, and activation is blocked by both inhibitors. If we assume
that PMA is indeed specific for the conventional isoforms of PKC, then the
implication is that one of those isoforms is also involved in the upstream
regulation of the pathway. It is not without precedent for one isoform of PKC
to regulate the activity of another; for example, PKC
seems to be
involved in the regulation of novel PKC isoforms
(12).
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DISCUSSION |
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The use of antibodies against relatively short peptide sequences of
signaling proteins is widespread. Our data emphasize that even with antibodies
to overlapping sequences, use of antibody to just one sequence may not reveal
significant aspects of signaling events. This is particularly the case for PKC
II, where multiple changes in conformational states occur in relation to
changes in phosphorylation at multiple sites. Thus, Newton and co-workers
(15), using
baculovirus-expressed PKC
II in vitro, have provided extensive
data supporting a model in which global conformational changes following
phosphorylation result in masking and unmasking of the C-terminal region. Such
changes can result in antibody binding being blocked, either directly because
of phosphorylation within the target sequence of the antibody or because of
the masking of different sections of the sequence as a result of
conformational changes. One possible explanation of the data, therefore, is
that more subtle conformational changes may also occur within the C-terminal
region that lead to different parts being exposed or masked in different
species of PKC
II, leading to differential recognition by the two
antibodies.
Physiological Significance of Phosphorylation and Turnover of PKC
II for Intestinal Sugar AbsorptionWe have previously
reported that sugar absorption in rat small intestine is up-regulated by
activation of PKC
II favoring insertion of GLUT2 into the brush-border
membrane (2,
7). Up-regulation is blocked by
chelerythrine or Ro 31-8220, which acts by binding to the catalytic site of
PKC
II. We have now shown that the inhibitors rapamycin and wortmannin,
which inhibit at different points in the signaling pathway controlling PKC
maturation, have the same effects on absorption and GLUT2 trafficking. The
data therefore provide an independent confirmation in scientific terms of our
previous work on which the GLUT2 model of rapid regulation of intestinal sugar
absorption was based (1).
Our data suggest that the signaling pathway controlling the involvement of
PKC II in the regulation of intestinal sugar absorption comprises two
main parts: phosphorylation of PKC
II is followed by cleavage, turnover,
and degradation (Fig. 7). Phosphorylation of Thr-500 in the activation loop by PDK-1 downstream of
PI3-kinase is followed by autophosphorylation of Thr-641 and Ser-660 in the
C-terminal sequence
(1315,
25); PI3-kinase-independent
mechanisms may also exist in some circumstances
(28). The formation of
competent PKC
II is associated in intestine with binding to the
brush-border membrane and substantial cleavage of native enzyme to a 49-kDa
catalytic domain. Activation of PKC
II by PMA at the brush-border
membrane favors insertion of GLUT2 into the brush border and so increases
sugar absorption; further cleavage also occurs, increasing the level of the
49-kDa species at the brush-border membrane (Figs.
1 and
2)
(7). Since the intestinal
absorptive cell is polarized, it is supplied with low concentrations of
glucose from the blood. Yet luminal perfusion with Krebs-Henesleit buffer
alone does not promote further cleavage and turnover, whereas perfusion with 5
mM fructose (Figs. 2
and 5) or low concentrations of
glucose (2) does. It therefore
seems that there must be an additional sensing mechanism at the brush-border
membrane by which sugars supplied in the lumen activate PKC
II
(Fig. 7); such a mechanism
presumably involves increases in cytosolic Ca2+ and
diacylglycerol (1,
2). As noted, mannitol promotes
turnover and degradation but is not transported. Whether there is a specific
sugar sensor is open to debate, but it is of interest to note that SGLT3,
which binds but does not transport glucose, can depolarize membranes in which
it resides. Luminal sugars promote rapid turnover of PKC
II by
dephosphorylation of the 49-kDa species to the 42-kDa species followed by
polyubiquitylation to the 180- and 250+-kDa species prior to degradation at
the proteasome.
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On the basis of these observations, we can envisage a possible overall
mechanism for the regulation of sugar absorption. In perfusions with fed
animals, but when no sugar is present in the lumen at the start of an
experiment, plasma sugar and insulin levels are normal. The amount of
competent PKC II available for activation is determined by the
prevailing balance between its rate of formation by phosphorylation and the
rate of cleavage, turnover, and degradation. Luminal perfusion of sugars
causes translocation of competent PKC
II to the brush-border membrane
through a brush border-sensing mechanism, resulting in activation, cleavage,
and turnover and degradation. The balance of the different pathways is
different with different sugars so that glucose and fructose promote the
49-kDa form of PKC
II and favor insertion of GLUT2 into the membrane as
compared with mannitol (Fig.
5). Moreover, a high level of glucose favors rapid insertion of
GLUT2 as compared with a low level of glucose (Fig. 3 of Ref.
2). Increased insertion of
GLUT2 into the membrane results in increased absorption and therefore
transintestinal delivery of sugars to the circulation; as plasma sugar levels
increase, release of insulin is stimulated, activating PI3-kinase and thereby
PDK-1 to increase the rate of formation of competent PKC
II. Amino acids
have a similar, although less potent, effect on insulin release. Moreover, an
amino acid-sensing pathway also promotes the formation of competent enzyme by
activating mTOR, which is thought to prevent dephosphorylation of the
hydrophobic site Ser-660, which is crucial for PKC
II activity, by
inhibiting a phosphatase (12,
18,
19,
29). Thus, perfusion of
jejunum with either wortmannin or rapamycin in the presence of fructose blocks
the formation of competent PKC
II and the whole of the signaling pathway
(Figs. 2 and
6). As absorption proceeds, the
fall in luminal sugar concentrations both inactivates PKC
II and
ultimately diminishes plasma sugar and insulin levels, by which time the
initial level of competent PKC
II is restored by re-establishment of the
balance between the rates of formation and cleavage, turnover, and
degradation. This results in loss of GLUT2 from the brush-border membrane and
down-regulation of sugar absorption. Wortmannin and rapamycin by inhibiting
the formation of competent PKC
II alter the balance strongly in favor of
turnover and degradation. It therefore appears that the control of PKC
II phosphorylation has the potential to provide a mechanism for the
observation that intestinal sugar absorption is regulated by amino acids
(5) and insulin
(20,
3032).
The PKC II turnover and degradation pathway appears to be strongly
emphasized in intestine. Moreover, the degradation of PKC
II is much
faster than that of PKC
(24), which is also present in
intestine (33), so that PKC
II seems more suited to the short term control of sugar absorption than
PKC
. The significance of our findings for the regulation of intestinal
sugar transport is therefore clear. Immediately after a meal, the absorptive
capacity of the brush-border membrane is rapidly up-regulated to match dietary
intake as a large local concentration of glucose is generated at the surface
of the brush-border membrane by the hydrolysis of luminal disaccharides and
-limit dextrins so that PKC
II is rapidly activated and GLUT2 is
inserted into the membrane (1).
As sugars are absorbed and luminal concentrations decrease, so PKC
II is
inactivated, and the GLUT2-mediated component is down-regulated by the
inactivation and loss of GLUT2 from the membrane. Blood sugar and insulin
levels peak within 1 h, returning to normal after about 2 h; the initial
events controlling intestinal delivery of sugars occur on a time scale of
minutes. Such rapid up- and down-regulation requires the dynamic control
afforded by the rapid turnover and degradation of PKC
II provoked by
dietary sugars.
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FOOTNOTES |
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To whom correspondence should be addressed. E-mail:
glk1{at}york.ac.uk.
1 The abbreviations used are: PKC, protein kinase C; PKB, protein kinase B;
PI3-kinase, phosphatidylinositol 3-kinase; mTOR, mammalian target of
rapamycin-dependent; PMA, phorbol 12-myristate 13-acetate.
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REFERENCES |
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