(Received for publication, October 17, 1995; and in revised form, January 23, 1996)
From the
D-chiro-Inositol is an epimer of myo-inositol that is found in certain mammalian
glycosylphosphatidylinositol protein anchors and inositol
phosphoglycans possessing insulin-like bioactivity. In order to
generate a probe for metabolic studies, D-chiro-[3-H]inositol was
synthesized by selective reduction of D-chiro-3-inosose at pH 6.5 with sodium borotritide. D-chiro-[3-
H]Inositol was taken
up by HepG2 human liver cells through a saturable and stereospecific
pathway in which D-chiro-inositol and myo-inositol competed equally but L-chiro-inositol was not recognized. D-Glucose, but not L-glucose, competed for D-chiro-[3-
H]inositol uptake
over glucose concentrations of 4-28 mM. Maximum
transport capacity was 717 pmol/mg cell protein/3 h with a K
value of 348 µM. Uptake
was reduced by 76% when sodium was eliminated from the medium and by
94% when the experiment was performed at 0 °C.
The new myo/D-chiro-inositol transporter is distinct from the sodium-myo-inositol co-transporter found in many tissues and accounts for all of the saturable D-chiro-inositol uptake and for a portion of the saturable low affinity myo-inositol uptake in HepG2 cells. It may allow D-chiro-inositol to be used by cells in the presence of a relatively large amount of competing myo-inositol.
myo-Inositol, one of nine isomers of hexahydroxycyclohexane, comprises almost all of the naturally occurring inositol of mammalian tissues. However, a small amount of chiro-inositol, an optically active epimer of myo-inositol, is also present and may have physiological importance. D-chiro-Inositol was recently found unexpectedly in inositol phosphoglycans thought to be mediators of insulin signaling (1, 2) and in glycosylphosphatidylinositol protein anchors(3, 4) . The related compound pinitol, 3-O-methyl-D-chiro-inositol, is a prominent component of dietary legumes such as soybeans(5) . Clinical studies have demonstrated that diabetic patients excrete large amounts of D-chiro-inositol in urine related to the degree of diabetic glycemic control(6, 7) , although the opposite result has also been reported(8) . Treatment of diabetics with insulin resulted in decreased urinary excretion and transient elevation of plasma D-chiro-inositol(6) . These experiments show that D-chiro-inositol and related substances are recognized by cell systems, but little is known about the biochemical pathways involved.
To allow further study of this unusual cyclitol
with high sensitivity we have prepared radiolabeled D-chiro-inositol by reduction of D-chiro-3-inosose using sodium borotritide. The
reaction product, D-chiro-[3-H]inositol, is taken
up avidly by cultured HepG2 liver cells in a pathway that is
stereospecific and shared with myo-inositol.
For GC/MS analysis inositol samples were derivatized by
incubating with 10% pentafluoropropionic anhydride in acetonitrile at
65 °C for 30 min. The derivatizing reagent was evaporated almost to
dryness, and the samples were reconstituted in acetonitrile and
injected onto a 25 0.32-mm inner diameter Chirasil-Val fused
silica capillary column with 0.2-µm film thickness (Alltech
Associates, Deerfield, IL) and analyzed on an HP 5970 electron impact
ionization positive ion mass spectrometer. A temperature program of 100
°C for 1 min followed by a programmed rise to 130 °C at 5
°C/min was used. Ions at m/z 565 and 569, which
are characteristic of natural and hexadeuterated derivatized inositols,
respectively, were monitored. This chromatographic technique resolves D and L enantiomers of chiro-inositol(6) .
Figure 1: Preparation of labeled D-chiro-inositol.
Figure 2: Reaction products from reduction of D-chiro-3-inosose. A, GC/MS tracing showing approximately equal amounts of the pentafluoropropionyl derivatives of D-chiro-inositol and allo-inositol using a Chirasil-Val chiral column. The retention time of myo-inositol is shown to demonstrate the absence of myo-inositol in the reaction products. The ion intensities of D-chiro-inositol and allo-inositol under these conditions expressed as areas are equal (ratio 1.01). B, GC/MS tracing of reaction product from A with an expanded time scale. C, GC/MS tracing of racemic hexadeuterated chiro-inositol internal standard showing separation of D and L enantiomers.
HPLC-purified D-chiro-3-inosose (5 mg) was then reduced with sodium borotritide as described under ``Experimental Procedures'' and purified by high voltage preparative paper electrophoresis. The product migrating in the position of chiro-inositol contained 100 mCi of tritium and was homogeneous, comigrating with authentic chiro-inositol on silica gel G TLC in 10:3 acetone:water (data not shown) and on HPLC with 76:24 acetonitrile:water as mobile phase (Fig. 3). By HPLC the preparation was free of radiolabeled myo-inositol and allo-inositol (elution positions marked with arrows in Fig. 3). This material was used for further studies.
Figure 3:
Purity of D-chiro-[H]inositol. A sample
consisting of 20 µl of a 20 mg/ml solution of authentic chiro-inositol containing 2000 dpm of D-chiro-[3-
H]inositol was
chromatographed on aminopropyl-silica in 76:24 acetonitrile:water with
monitoring of the optical density at 206 nm and radioactivity present
in 0.5-1-ml fractions. The optical density peak at 4 ml is an
injection artifact.
Figure 4:
Uptake of D-chiro-[H]inositol by HepG2
cells. Cells were washed twice with warm PBS and incubated at 37 °C
for the indicated times with PBS containing 1.5 µCi/ml D-chiro-[3-
H]inositol. At the
end of the experiment cells were washed 4 times with cold PBS and
solubilized in 0.1 N NaOH. The open circle depicts an
identical experiment in which the cells were incubated with D-chiro-[3-
H]inositol at 0
°C rather than 37 °C. Error bars represent standard
deviation of triplicate wells and where missing are within the symbol.
Very little of the cell-associated tracer was found in phospholipids or proteins after 3 h of incubation. Cells from an experiment similar to that shown in Fig. 4were extracted with 1.5 ml of 1:2 chloroform:methanol containing 0.05 N HCl for 30 min at room temperature, and the insoluble protein pellet was removed by centrifugation. Then 0.5 ml of chloroform and 0.9 ml of water were added, and the organic and aqueous phases were separated, dried, and counted. The aqueous phase contained 95.6 ± 3.5% of the recovered DPM, the organic phase contained 0.35 ± 0.06%, and the protein pellet contained 4.0 ± 0.7%.
The specificity of D-chiro-[3-H]inositol transport
is shown in Fig. 5. For Fig. 5A HepG2 cells were
incubated with D-chiro-[3-
H]inositol with and
without 1 mg/ml of competing unlabeled inositol. D-chiro-Inositol displaced 43% of the radioactive
tracer (p < 0.0001), and pinitol (the structurally related
3-O-methyl-D-chiro-inositol found in
soybeans) displaced 27% of the tracer (p < 0.0001).
However, L-chiro-inositol was ineffective, the tracer
uptake remaining 99.2 ± 1.4% of the control. Fig. 5B shows that D-glucose reduced tracer uptake progressively
over the concentration range of 4-28 mM (p = 0.0087 for an effect of glucose by analysis of variance).
However, L-glucose showed no tracer displacement even at 28
mM, and tracer uptake remained 96.4 ± 5.5% of the
control (p = 0.67).
Figure 5:
Specificity of D-chiro-[H]inositol transport
in HepG2 cells. 1.5 µCi/ml tracer in PBS either without addition (control) or with competitor was incubated with cells for 3 h
at 37 °C. The cells were then washed and dissolved in sodium
hydroxide. A, competition by 5.6 mMchiro-inositol enantiomers or 5.2 mM pinitol. B, competition by glucose enantiomers. Error bars depict the S.E. of triplicate wells.
Fig. 6gives kinetics of D-chiro-inositol transport by HepG2 cells. Tracer and
unlabeled D-chiro-inositol were mixed in PBS and
incubated with HepG2 cells for 3 h. Fig. 6A shows that
5.6 mM unlabeled D-chiro-inositol displaced
63.7% of the radioactive tracer. The Eadie-Hofstee plot of specific
binding in Fig. 6B is linear with a computed K = 348 µM and maximum uptake
capacity of 717 pmol/mg.
Figure 6:
Kinetics of D-chiro-[H]inositol. HepG2
cells were incubated for 3 h in PBS containing 1.0 µCi/ml tracer
and various concentrations of unlabeled D-chiro-inositol. A, displacement of tracer
by unlabeled D-chiro-inositol. Points represent
triplicate wells ± S.E., and error bars where missing
are within the symbol. B, Eadie-Hofstee plot of the
data of Fig. 6A after subtracting the nonspecific
uptake observed in the presence of 5.6 mMD-chiro-inositol.
D-chiro-[3-H]Inositol
transport was dependent upon extracellular sodium ion. After replacing
the sodium chloride and sodium phosphate in PBS by lithium salts the
uptake of 1 µCi/ml D-chiro-[3-
H]inositol in HepG2
cells over 3 h was reduced by 73.4% from 8823 ± 129 dpm/mg to
2347 ± 78.3 dpm/mg (p < 0.0001 for triplicate
wells). For these experiments all cell washing both before and after
tracer binding was done in sodium-free PBS.
The critical question of
the relation between D-chiro-inositol uptake and myo-inositol uptake was studied in the following experiments.
First, as seen in Fig. 7, unlabeled myo-inositol and D-chiro-inositol appeared to compete equally for
uptake of D-chiro-[3-H]inositol
tracer in HepG2 cells (p = 0.18 for a difference
between the two inositols by 2-way analysis of variance). However, when myo-[2-
H]inositol was used as a tracer
instead a much different picture emerged, as seen in Fig. 8.
Here myo-inositol was a much more potent competitor, reducing
tracer binding by 78% at a concentration of 0.17 mM where no
corresponding effect of D-chiro-inositol was
observed. However, at higher concentrations of competitors there was
clearly some incomplete displacement of the myo-[
H]inositol tracer. Thus, the two
inositol tracers appeared to differ kinetically. Fig. 8B gives an Eadie-Hofstee semireciprocal plot of the displacement of myo-[
H]inos
itol tracer by unlabeled myo-inositol after subtraction of nonsaturable uptake. A
curvilinear relation is seen. When the uptake of Fig. 8B was corrected by subtracting myo-inositol uptake
attributable to the myo-inositol/D-chiro-inositol transport
pathway of Fig. 7(see Fig. 8legend), the plot became
linear with K
= 37.5 µM and
maximum uptake capacity of 1240 pmol/mg.
Figure 7:
Competition of myo-inositol for D-chiro-[H]inositol uptake.
HepG2 cells were incubated for 3 h at 37 °C with 1 µCi/ml D-chiro-[3-
H]inositol tracer
plus the indicated concentrations of unlabeled inositols. Triplicate
wells were analyzed.
Figure 8:
Kinetics of myo-[H]inositol uptake. A,
HepG2 cells were incubated with 1 µCi/ml myo-[2-
H]inositol in PBS in the presence
of unlabeled inositols. Values are for triplicate wells. B,
Eadie-Hofstee plot of the unlabeled myo-inositol displacement curve of panel A after subtraction of the nonspecific uptake observed
in the presence of 5.6 mMmyo-inositol. C,
same as part B after further subtraction of the uptake attributable to
the myo-inositol/D-chiro-inositol uptake
pathway. The latter was computed as the displacement of tracer by D-chiro-inositol divided by the displacement of
tracer by myo-inositol multiplied by the total myo-inositol mass uptake.
The uptake of myo-[H]inositol differed from that of D-chiro-[3-
H]inositol in
the
stereospecific discrimination of glucose (Table 1). Both D-glucose and L-glucose were recognized by the myo-[
H]inositol uptake pathway and
produced 55 and 45% reduction in tracer uptake, respectively.
The
relation of the myo-/D-chiro-inositol
transporter to facilitated glucose transporters and to sodium-dependent
sugar co-transporters was examined by performing D-chiro-inositol uptake studies in the presence and
absence of cytochalasin B, which binds to and inhibits facilitated
glucose transporters, and phlorizin, which does not affect facilitated
glucose transporters but inhibits sodium-dependent transporters (Fig. 9). Saturable D-chiro-[3-H]inositol uptake
was not affected significantly by cytochalasin B but was reduced 93% by
phlorizin (p < 0.0001).
Figure 9:
Effect of inhibitors on saturable D-chiro-[H]inositol uptake. Left, triplicate wells of HepG2 cells were incubated as
described under ``Experimental Procedures'' with PBS
containing either 0.1% ethanol (control) or 0.1% ethanol plus 80
µM cytochalasin B. Right, cells were incubated
with PBS containing no addition (control) or 1 mM phlorizin.
Nonsaturable uptake observed in the presence of 5.6 mM unlabeled D-chiro-inositol under the same
conditions was subtracted from the total uptake to give saturable
uptake. Error bars depict S.D.
Uptake of D-chiro-[3-H]inositol was also
studied in the IMR-90 diploid human fibroblast strain. The amount of
uptake observed during a 3-h incubation at 37 °C using 1.0
µCi/ml tracer was 1897 ± 125 dpm/mg cell protein, 25% of the
value for HepG2 cells. Uptake was not affected by 1 mg/ml L-chiro-inositol but was significantly reduced by 24%
with 1 mg/ml D-chiro-inositol (p =
0.04) and by 49% with 1 mg/ml myo-inositol (p = 0.003). Therefore, a pathway for D-chiro-inositol uptake appears to be present in
these cells but is expressed at a lower level than in HepG2 cells.
The synthesis of a radioactive D-chiro-inositol tracer should facilitate studies of D-chiro-inositol-containing structural and regulatory molecules in mammalian cells. Tritium labeling of D-chiro-inositol was achieved by borotritide reduction of D-chiro-3-inosose (Fig. 1). The yield of D-chiro-inositol as a percentage of the inositol reaction product was increased to 50% from about 10% as previously reported (12) by performing the reaction at pH 6.5 in phosphate buffer, as determined in nonradioactive preparations. In a radioactive preparation after purification by paper electrophoresis, the final tracer was analyzed by HPLC on aminopropyl-silica in an acetonitrile:water solvent system, which gave good resolution of various inositols and inososes and showed that the final product was pure (Fig. 3). GC/MS analysis of a similar unlabeled reaction product showed that only the D enantiomer of chiro-inositol was produced (Fig. 2).
D-chiro-[3-H]Inositol tracer
appears to be taken up by HepG2 liver cells through a novel pathway for
which we suggest the name myo/D-chiro transporter. myo-Inositol and D-chiro-inositol are recognized equally (Fig. 7), but uptake is highly stereospecific in that L-chiro-inositol is not recogniz
ed at all (Fig. 5A). This enantiospecificity implies that the
transporter is also asymmetric with a defined spatial orientation, as
would be expected in a transport protein. The myo/D-chiro transporter shares some features
of the well-studied transporter for myo-inositol, which is
found in many cells and
tissues(14, 15, 16, 17, 18) .
For example, it requires the extracellular sodium ion and has
appreciable cross-competition with D-glucose (Fig. 5B). Glucose over a range of 4 mM (similar to fasting plasma concentrations) to 28 mM (found in very poorly controlled diabetic patients) reduces D-chiro-inositol
uptake in cultured liver cells. This
provides a presumptive mechanism for the observation that diabetic
subjects excrete D-chiro-inositol in the urine in
proportion the degree of hyperglycemia present(6) . In the
kidney excessive glucose might interfere competitively with D-chiro-inositol reabsorption and increase the amount
excreted. It also suggests the possibility that the presence of
hyperglycemia might interfere with tissue uptake of D-chiro-inositol elsewhere in the body and reduce
intracellular D-chiro-inositol stores.
However,
the myo/D-chiro transporter differs from the myo-inositol transporter in several respects. First, the two
inositols compete equally in the former but D-chiro-inositol competes poorly if at all in the
latter (Fig. 8A versusFig. 7). Second, L-glucose is not recognized by the myo/D-chiro transporter (Fig. 5B), but it competes almost as well as D-glucos
e in the myo-inositol pathway (Table 1). The lack of stereospecificity for glucose in the myo-inositol transporter that we observed confirms a previous
report in which the two glucose enantiomers were equally competitive in
reducing H-myo-inositol uptake in endothelial
cells (17) . Finally, the K
values of the
two transporters for myo-inositol are markedly different. In
our hands the K
for myo-inositol of the myo-inositol transporter is 43.4 µM if
uncorrected data are used and 37.5 µM if the corrections
detailed in the legend to Fig. 8are performed. Both of these
values are close to values of 12-41 µM reported
previously for high affinity uptake in cultured cells (16, 17) and are also close to the reported human
plasma free myo-inositol concentration of 24.5
µM(6) . In contrast, the K
of
the myo/D-chiro transporter for both
inositols is 348 µM. Thus, this pathway would not be
saturated at normal plasma concentrations of myo-inositol and
would allow uptake of both inositols under physiological conditions.
The myo/D-chiro transporter appears to be
unrelated to facilitated glucose transporters because it is
sodium-requiring, phlorizin-inhibitable, and unaffected by cytochalasin
B(19) .
Our data show that HepG2 cells, which are
transformed cells derived from liver, have a high affinity saturable
transporter for myo-inositol (Fig. 8). Previous studies
of dissociated normal liver cells have described only pathways for myo-inositol uptake that are poorly saturable or nonsaturable
at millimolar levels of myo-inositol(20, 21, 22) . However,
in that work the ability of unlabeled myo-inositol to displace
trace amounts of H-myo-inositol was not reported
explicitly, and it is possible that a component of high affinity uptake
was also present. myo-Inositol transport pathways in liver
require further study. Fig. 6A and Fig. 7show
that nonsaturable pathways (or pathways with K
values much higher than 0.348 mM) account for a portion
of the uptake of D-chiro-inositol, and it is possible
that this uptake might be similar to the nonsaturable myo-inositol pathway of liver.
Our findings may have
implications with respect to the transport of myo-inositol as
well as D-chiro-inositol. As discussed above,
previous reports have shown that tissues and cells have high affinity
saturable myo-inositol uptake (K of about
30 µM) and nonsaturable uptake that is apparently
carrier-mediated. But in addition, studies in which radioactive tracer
was displaced by unlabeled competitor uniformly have shown a third
pathway consisting of saturable sites of apparently lower affinity,
which are frequently quite prominent and are recognized by nonlinear
Eadie-Hofstee plots(15, 16) . These sites are absent
in oocytes injected with myo-inositol transporter
transcripts(18) . Our data suggest that at least part of this
low affinity saturable pathway is the myo/D-chiro transporter (Fig. 8). The Eadie-Hofstee plot of the
displacement of
H-myo-inositol tracer by unlabeled myo-inositol is curved even though nonsaturable binding has
been subtracted, suggesting that two saturable uptake sites are present (Fig. 8B). Although it is theoretically possible to
deconvolute this curve into two sites mathematically, this is not
practical because of an inherent lack of precision in estimating the
parameters of the two sites, as noted previously with kidney cells (13) . However, by considering the additional information
contained in the experiment shown in Fig. 8in which
displacement of
H-myo-inositol tracer by both myo-inositol and D-chiro-inositol was done,
it is possible to improve estimation of the high affinity myo-inositol uptake pathway. In the resulting analysis we made
two simplifying assumptions: 1) both inositols compete equally for the myo/D-chiro transporter (see Fig. 7);
2) only myo-inositol is recognized by the high affinity myo-inositol transporter (see Fig. 8A). When
the cell myo-inositol uptake was corrected for the amount
passing through the lower affinity myo/D-chiro transporter the Eadie-Hofstee plot became linear (Fig. 8, B and C). This analysis suggests that at least some
of the saturable but low affinity myo-inositol uptake observed
in HepG2 cells can be accounted for by the myo/D-chiro transporter.
These results
show that an alternative pathway exists for inositol transport in
cultured cells, which can be revealed by use of radiolabeled D-chiro-[3-H]inositol. Further
studies are needed to evaluate the physiological significance, tissue
specificity, and potential importance of this pathway in the regulation
of cell signaling and protein structure.