![Glucosamine decreases [ 3 H]-2-DG uptake and glucose consumption. A. Confluent VSMC were exposed to media containing 5.6 mmol/L glucose with the indicated concentrations of glucosamine (n ¼ 3). After 24 hours in the indicated conditions cells were exposed to 0.2 mCi of [ 3 H]-2-DG for 15 minutes and [ 3 H]-2-DG uptake was determined as described in the Methods section. Values are expressed relative to total cell protein as mean + SEM. *P 5 0.01 as determined by multiple pair-wise comparisons. B. Confluent VSMC were exposed to media containing 5.6 mmol/L glucose with the indicated concentrations of glucosamine for 24 hours (n ¼ 3). Media glucose content was determined after 24 hours in the indicated conditions, and was subtracted from baseline glucose content (5.6 mmol/L or 25 mmol/L). Values are expressed as mean + SEM. *P 5 0.05 as determined by multiple pair-wise comparisons.](https://www.researchgate.net/profile/Peter-Little-2/publication/5366913/figure/fig1/AS:433639772168193@1480399187166/Glucosamine-decreases-3-H-2-DG-uptake-and-glucose-consumption-A-Confluent-VSMC-were_Q320.jpg)
Content uploaded by Peter J Little
Author content
All content in this area was uploaded by Peter J Little on Nov 29, 2016
Content may be subject to copyright.
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=iarp20
Download by: [UQ Library] Date: 28 November 2016, At: 21:57
Archives of Physiology and Biochemistry
ISSN: 1381-3455 (Print) 1744-4160 (Online) Journal homepage: http://www.tandfonline.com/loi/iarp20
Glucosamine inhibits the synthesis of
glycosaminoglycan chains on vascular smooth
muscle cell proteoglycans by depletion of ATP
Peter J. Little, Katherine D. Drennon & Lisa R. Tannock
To cite this article: Peter J. Little, Katherine D. Drennon & Lisa R. Tannock (2008) Glucosamine
inhibits the synthesis of glycosaminoglycan chains on vascular smooth muscle cell
proteoglycans by depletion of ATP, Archives of Physiology and Biochemistry, 114:2, 120-126,
DOI: 10.1080/13813450802033909
To link to this article: http://dx.doi.org/10.1080/13813450802033909
Published online: 10 Oct 2008.
Submit your article to this journal
Article views: 44
View related articles
ORIGINAL ARTICLE
Glucosamine inhibits the synthesis of glycosaminoglycan chains on
vascular smooth muscle cell proteoglycans by depletion of ATP
PETER J. LITTLE
1,2
, KATHERINE D. DRENNON
3
, & LISA R. TANNOCK
3,4
1
Baker Heart Research Institute, Cell Biology of Diabetes Laboratory, Melbourne, VIC, 3004, Australia,
2
Monash University
School of Medicine (Alfred Hospital), Departments of Medicine and Immunology, Prahran VIC, 3004, Australia,
3
Division of
Endocrinology and Metabolism, University of Kentucky, Lexington, KY, USA, and
4
Department of Veterans Affairs Medical
Center, Lexington, KY, USA
Abstract
Glucosamine via GlcNAc is a precursor for the synthesis of glycosaminoglycan (GAG) chains on proteoglycans. We
previously found that proteoglycans synthesized and secreted by vascular smooth muscle cells (VSMC) in the presence of
supplementary glucosamine had GAG of decreased not increased size. We investigated the possibility that the inhibition of
GAG chains synthesis on proteoglycans might be related to cellular ATP depletion. Confluent primate VSMCs were exposed
to glucosamine, azide, or 2-deoxyglucose (2-DG). Each of these agents depleted cell ATP content by 25–30%. All agents
decreased
35
S-SO
4
incorporation and reduced the size of the proteoglycans, decorin and biglycan as assessed by SDS-PAGE.
On withdrawal of the glucosamine, azide or 2-DG ATP levels and proteoglycan synthesis returned towards baseline values.
Glucosamine decreased glucose uptake and consumption suggesting that ATP depletion was due preferential
phosphorylation of glucosamine over glucose. Thus, glucosamine inhibition of proteoglycan synthesis is due, at least in
part, to depletion of cellular ATP content.
Key words: Glucosamine, proteoglycan synthesis, glycosaminoglycan, ATP depletion.
Introduction
Proteoglycans are a ubiquitous group of molecules
comprised of a core protein with one or more
glycosaminoglycan (GAG) side chains (Wight et al.,
1991; Hascall et al., 1991). Proteoglycans are
synthesized by the initial formation of the core
protein followed by the addition of a tetrasaccharide
linkage region to a serine residue on the core protein
and finally the extension of the GAG chains by the
sequential addition of monosaccharides with sulfa-
tion and epimerisation forming the mature GAG
chain (Wight et al., 1991). The substrates utilized in
GAG polymerization are GalNAc and GluA and the
GalNAc is derived from glucosamine (Wight et al.,
1991; Kitagawa et al., 2003).
Glucosamine is a component of the hexosamine
pathway (Marshall et al., 2004) a pathway by which
high glucose levels can alter cellular function leading
to the vascular complications of diabetes (Wells et al.,
2003; King et al., 1996). In the hexosamine pathway
an amide group is added to a glucose metabolite
leading to the formation of glucosamine, thus high
glucose levels increase cellular glucosamine levels. In
the context of studying the role of high glucose in
mediating the vascular complications of diabetes we
recently investigated the effect of glucosamine on
proteoglycan synthesis in VSMCs (Tannock et al.,
2002).
Glucosamine is a precursor for the synthesis of
GAG chains via its conversion to GalNAc (Hascall
et al., 1991). Thus, we previously hypothesized that
increased glucosamine availability would lead to
increased GAG synthesis and higher molecular
weight proteoglycans due to longer GAG chains
(Tannock et al., 2002). Elongation of GAG chains on
VSMC proteoglycans such as biglycan mediated by
growth factors such as transforming growth factor b
(Little et al., 2002) increases binding to low density
lipoproteins (LDL) and this is strongly implicated
with the retention of atherogenic lipoproteins in the
arterial wall and thus atherosclerosis (O’Brien et al.,
Correspondence: Lisa R. Tannock, Division of Endocrinology and Molecular Medicine, Department of Medicine, Room 567, Wethington Building, 900 S.
Limestone, University of Kentucky, Lexington, KY, 40536-0200, USA. Tel: 859-323-4933 ext. 81415. Fax: 859-257-3646. E-mail: Lisa.Tannock@uky.edu
Received for publication 3 December 2007. Accepted 31 January 2008.
Archives of Physiology and Biochemistry, April 2008; 114(2): 120 –126
ISSN 1381-3455 print/ISSN 1744-4160 online ª2008 Informa UK Ltd.
DOI: 10.1080/13813450802033909
1998; Nakashima et al., 2007; Skalen et al., 2002;
Ballinger et al., 2004). Surprisingly, we found that
VSMCs exposed to excess glucosamine in vitro
synthesized proteoglycans with shorter not longer
GAG chains (Tannock et al., 2002). The response
was concentration dependent up to a glucosamine
concentration of 32 mM. The response was also
functionally significant because the shorter GAG
chains lead to reduced binding of proteoglycans to
LDL as was demonstrated in the gel mobility shift
assay (Tannock et al., 2002; Camejo et al., 1993).
The mechanism(s) by which glucosamine de-
creases the GAG synthesis is not known. Glucosa-
mine enters cells through glucose transporters, and is
rapidly phosphorylated by hexokinase to GlcN-6-P
(GlcN-6-P) which consumes ATP and is then
converted via several steps to UDP-N-acetylglucosa-
mine (UDP-GlcNAc) (Marshall et al., 2004). Glu-
cosamine supplementation has been shown to
deplete cellular ATP levels in a variety of cell types
(Hresko et al., 1998; Kang et al., 2001; Han et al.,
2003; Anello et al., 2004; Marshall et al., 2004),
possibly by utilization of ATP derived phosphate as
glucosamine is converted into GlcN-6-P (Hresko
et al., 1998) similar to the mechanism by which 2-
deoxygluocse depletes cells of ATP (Little et al.,
1988). The present study was undertaken to address
the question if the effect of glucosamine to inhibit
VSMC GAG synthesis on proteoglycans is due to
intracellular ATP depletion. We demonstrate that
cellular ATP depletion is the mechanism by which
glucosamine exerts its unexpected action on GAG
synthesis in vascular smooth muscle.
Materials and methods
Chemicals and reagents
Chemicals and reagents were obtained from Sigma
(St. Louis, MO) unless otherwise specified.
Cell culture
Monkey (macaca nemestrina) VSMCs (a generous gift
from Dr. T. Wight, Hope Heart Institute, Seattle,
WA) were grown to confluence in 12-well cell culture
dishes in Dulbecco’s Modified Eagle Medium
(DMEM) containing 5.6 mmol/L glucose with 5%
serum, then were serum deprived in 0.1% for 48
hours. Quiescent cells were exposed to glucosamine
(0–32 mmol/L) in media containing either 5.6 mmol/
L or 25 mmol/L glucose, for 24 hours with 5% serum
as previously described (Tannock et al., 2002). Cells
were metabolically labeled with
35
SO
4
(75mCi/ml) for
24 hours. To evaluate the effect of ATP depletion on
proteoglycan synthesis parallel dishes were exposed
to sodium azide (5 mmol/L) or 2-deoxyglucose (2-
DG, 10 mmol/L) in DMEM containing 5.6 mmol/L
glucose. To evaluate the reversibility of the effects of
glucosamine, azide, or 2-DG in some experiments
cells were exposed to media containing 5.6 mmol/L
glucose +glucosamine, azide, or 2-DG for 24
hours, then changed to fresh media containing
5.6 mmol/L glucose alone with
35
S-SO
4
for a further
24 hours.
Proteoglycan isolation and analysis
Media from metabolically labelled cells was collected
for proteoglycan isolation and analysis as previously
described (Tannock et al., 2002).
35
S-SO
4
incorpora-
tion was determined using the cetyl pyridinium
chloride (CPC) method (Tannock et al., 2002).
Media was collected in the presence of protease
inhibitors and passed over DEAE-Sephacel mini-
columns. Secreted proteoglycans were analysed by
SDS-PAGE using a 4% to 12% gradient gel with a
3.5% stacking gel, and apparent molecular sizes were
estimated by comparing to
14
C molecular weight
markers (Amersham, Piscataway, NJ) (Schonherr
et al., 1991; Tannock et al., 2002). Lanes were loaded
with 25 000 dpm (
35
S) of total secreted proteogly-
cans synthesized in the presence of the indicated
agents.
Metabolic assays
After 24 hours media from each well was collected
for determination of glucose content with a commer-
cially available kit (Pointe Scientific, Inc, Canton,
MI). The cell layer was washed with phosphate
buffered saline (PBS), lysed with 0.1 N NaOH, and
aliquots were taken for determination of total cell
protein (DC Protein Assay Kit, BioRad Hercules,
CA) or ATP content (ATP Determination Kit,
Molecular Devices, Eugene, OR). The lactate
dehydrogenase activity of the medium was deter-
mined using a cytotoxicity detection kit (Boehringer
Mannheim, Indianapolis, IN). In parallel wells the
cell layer was washed three times with reaction buffer
(150 mM NaCl, 5 mM KCl, 1.2 mM MgSO
4
,
1.2 mM NaH
2
PO
4
, 10 mM HEPES, 0.1% BSA, pH
7.4), then exposed to 0.2 mCi/ml of (
3
H)- 2-
deoxyglucose ((
3
H)- 2-DG) in reaction buffer at
378C. After 15 minutes the plates were placed on ice
to halt the reaction, and the wells were washed four
times with cold PBS. The cell lysate from each well
was transferred to a scintillation vial and counted as a
measure of (
3
H)- 2-DG uptake.
Statistical analyses
Results are expressed as mean +standard error of
the mean (SEM). Data was analysed by one way
ANOVA where all comparisons are within media
containing 5.6 mmol/L glucose, or by two way
ANOVA evaluating the effect of media glucose
content and glucosamine, with multiple pair-wise
comparisons. Levels of statistical significance are
indicated in individual figures.
ATP depletion inhibits glycosaminoglycan synthesis 121
Results
Primate VSMCs were incubated with normal media
or high glucose-containing media with or without
glucosamine supplementation for 24 hours and the
cell ATP levels were assessed. Cell ATP levels were
decreased by 24% by the addition of glucosamine
(12 mM) to the normal media (P50.001,
Figure 1A). There was no difference in cell ATP
levels between normal and high glucose media, and
cell ATP levels were decreased by 30% by the
addition of glucosamine (12 mM) to the high glucose
media (P50.05; Figure 1A). The effect was depen-
dent on the dose of glucosamine (0–32 mM)
(P50.01, Figure 1B). Notably, the dose–response
relationship was almost identical to that reported
earlier by us for the glucosamine-mediated inhibition
of
35
S-SO
4
incorporation and the inhibition of GAG
elongation on proteoglycans secreted by VSMCs
(Tannock et al., 2002). The depletion of cell ATP
levels with glucosamine (16 mmol/L) had no effect
on total protein synthesis (range 0.16 +0.06 to
0.13 +0.06 mg/ml for VSMCs exposed to normal
media through glucosamine concentrations up to
16 mmol/L; P¼N.S.) or LDH activity (data not
shown). The effects of glucosamine supplementation
on cell ATP levels and on proteoglycan synthesis
were partially reversible. In VSMCs supplemented
with glucosamine for 24 hours, then incubated with
glucosamine-free media for a further 24 hours, ATP
levels returned towards baseline levels (P50.05)
(Figure 2A). Similarly,
35
S-SO
4
incorporation into
secreted proteoglycans (P50.001, Figure 2B) and
the effect on the size of the proteoglycans, decorin and
biglycan assessed by SDS-PAGE (Figure 2C) both
returned towards baseline levels on withdrawal of
glucosamine.
We next determined if depletion of cell ATP
content to a similar extent as that induced by
glucosamine but utilizing alternative biochemical
mechanisms of cellular ATP depletion would affect
VSMC proteoglycan synthesis. Sodium azide inhibits
mitochondrial ATP production and 2-DG consumes
ATP as it is metabolized to 2-deoxy-6-phosphate as it
enters the cell but in contrast to glucose-6-phosphate
the former is not a substrate for glycolysis (Hresko
et al., 1998; Vemuri et al., 1999; Kang et al., 2001).
Confluent quiescent VSMCs were exposed to
sodium azide (5 mmol/L), 2-DG (10 mmol/L) or
glucosamine (12 mmol/L) for 24 hours and cellular
ATP content and proteoglycan synthesis were
quantified. Azide, 2-DG and glucosamine all de-
creased cell ATP content to a similar extent com-
pared to unstimulated cells (P50.001, Figure 3A).
Treatment of cells with azide or 2-DG to reduce
cellular ATP levels mimicked the effect of glucosa-
mine to decrease
35
S-SO
4
incorporation into se-
creted proteoglycans (P50.01) (Figure 3B).
Furthermore, with this short term exposure neither
azide nor 2-DG were toxic to the cells as there was no
effect on total protein synthesis (data not shown). We
then assessed the reversibility of the response to ATP
depletion by azide and 2-DG on
35
S-SO
4
incorpora-
tion into proteoglycans and proteoglycan size as
assessed by SDS-PAGE. ATP levels (Figure 4A)
and
35
S-SO
4
incorporation into proteoglycans
(Figure 4B) all returned towards baseline levels. As
our interest was in GAG synthesis and structure we
assessed if the effect of ATP depletion on GAG
shortening was reversible. The
35
S-SO
4
labelled
proteoglycans were subjected to SDS-PAGE. Cell
ATP depletion inhibits GAG elongation leading to
lower molecular weight proteoglycans and this
response is partially reversible as evidenced by the
larger size of the proteoglycans, decorin and biglycan,
when the cellular ATP levels are partially replenished
(Figure 4C).
We investigated the mechanism by which glucosa-
mine depletes cells of ATP. Glucosamine enters cells
via glucose transporters but the rate limiting step is
the hexokinase mediated phosphorylation of gluco-
samine to GlcN-6-P, a step that utilizes ATP and
creates the high inward gradient for diffusion of
glucosamine into the cell (Harpur et al., 1949; Maley
et al., 1955). We assessed the acute (15 minute)
uptake of the glucose analogue 2-deoxyglucose
(2-DG) in the presence of various concentrations of
glucosamine. As the glucosamine concentration
increased the uptake of 2-DG was decreased in a
concentration dependent manner (Figure 5A). As
glucosamine appears to be preferentially taken up
and phosphorylated by hexokinase we assessed the
Figure 1. Glucosamine decreases cell ATP content. A. Confluent
VSMC were exposed to media containing either 5.6 mmol/L
glucose or 25 mmol/L glucose (solid bars) or also containing
12 mmol/L glucosamine (open bars) for 24 hours (n¼9). B.
Confluent VSMC were exposed to media containing 5.6 mmol/L
glucose with the indicated concentrations of glucosamine for
24 hours (n¼4). Cell ATP content was determined as described in
methods, and is expressed as mean +SEM relative to cells
exposed to media with 5.6 mmol/L glucose only, which is
expressed as 100%. *P50.001.
122 P. J. Little et al.
effects of glucosamine on rate of glucose utilization
from the culture media. With increasing concentra-
tions of glucosamine the rate of glucose consump-
tion from the media was progressively decreased
(Figure 5B). These data demonstrate that the
preferential use of ATP to phosphorylate glucosa-
mine instead of glucose leads to cellular ATP
consumption because glucose-6-phosphate is a sub-
strate for glycolysis and oxidative phosphorylation,
producing more ATP but glucosamine does not lead
to ATP production but rather an increased supply of
UDP-GlcNAc (Marshall et al., 2004).
Discussion
We previously observed, somewhat surprisingly, that
supplementing VSMC cultures with glucosamine,
the metabolic precursor of UDP-GlcNAc and thus of
GAG chains on proteoglycans, lead to the synthesis
of shorter and not longer GAG chains (Tannock
et al., 2002). In the current study we have investi-
gated the mechanism by which glucosamine leads to
inhibition of GAG chain elongation. We demonstrate
that glucosamine depletes cells of ATP and that two
alternative biochemical mechanisms of ATP deple-
tion, inhibition of oxidative phosphorylation and
glycolysis, also lead to inhibition of GAG elongation
on proteoglycans secreted by VSMCs. The effects of
ATP depletion are at least partially reversible as
Figure 3. ATP depletion decreases proteoglycan sulphate
incorporation and size. Confluent VSMC were exposed to media
containing either 5.6 mmol/L glucose alone (NG) or also
containing glucosamine (12 mmol/L), azide (5 mmol/L) or 2-
deoxyglucose (2-DG; 10 mmol/L) in the presence of
35
S-SO
4
(75mCi/ml) for 24 hours. A. Cell ATP content was determined as
described in methods (n¼6). B. Sulphate incorporation was
determined by CPC precipitation as described in methods (n¼6).
Data is expressed as mean +SEM relative to cells exposed to
media with 5.6 mmol/L glucose only which is expressed as 100%.
Figure 2. The effects of glucosamine are partially reversible. Cells were exposed to media containing 5.6 mmol/L glucose alone (NG) or
containing 12 mmol/L glucosamine (Glcs) as indicated for 24 hours, then all cells were cultured with fresh media as indicated containing
35
S-
SO
4
for a second 24-hour period (n¼7). A. Relative ATP content was determined as described in methods. B. Relative sulphate
incorporation was determined by CPC precipitation as described in methods. Data is expressed as mean +SEM relative to cells exposed to
media with 5.6 mmol/L glucose only for both 24-hour intervals, which is expressed as 100%. *P50.05 as determined by multiple pair-wise
comparisons. C. Proteoglycan size as determined by SDS-PAGE on 4–12% gradient gel with 3.5% stacking gel, with
14
C-labelled molecular
weight standards. Each lane was loaded with 25 000 dpm. The gel shown is representative of seven separate experiments.
ATP depletion inhibits glycosaminoglycan synthesis 123
partial restoration of ATP levels leads to partial
recovery of GAG synthesis. We further showed that
glucosamine depletes cells of ATP by competition for
glucose entry and hexokinase phosphorylation of
glucose. Thus, our data suggests that the mechanism
by which glucosamine decreases proteoglycan sul-
phate incorporation and size is via depletion of
cellular ATP content.
The data suggests that glucosamine supplementa-
tion leads to the preferential formation of GlcN-6-P
over Glu-6-P. GlcN-6-P leads to the increased
formation of UDP-GlcNAc which is a precursor for
GAG chain elongation on VSMCs so clearly the
effect of enhanced UDP-GlcNAc production to lead
to the synthesis of longer GAG chains is over-
whelmed by the effect of cellular ATP depletion to
inhibit GAG chain elongation.
Glucosamine supplementation has been shown to
deplete intracellular ATP in a number of cell types
including pancreatic beta cells (Anello et al., 2004)
rat skeletal muscle (Han et al., 2003), adipocytes
(Hresko et al., 1998) and human hepatoma cells
(Chang et al., 2003). Hresko et al. (1998) propose
that the ATP is depleted secondary to rapid
phosphorylation of glucosamine, and thus suggest
that glucosamine acts via a different mechanism
(ATP depletion) than high glucose, accounting for
some of the observed differences in effects induced
by glucosamine compared to high glucose. However,
in other cell types ATP depletion by exposure to
sodium azide or dinitrophenol did not mimic the
effects of glucosamine to induce insulin resistance
(Kang et al., 2001), thus ATP depletion is not the
sole mechanism underlying all of the effects of
glucosamine. Further studies are required to discern
Figure 4. The effects of ATP depletion on proteoglycan synthesis
are partially reversible. Cells were exposed to media containing
5.6 mmol/L glucose alone (NG) or containing azide (5 mmol/L) or
2-DG (10 mmol/L) as indicated for 24 hours, then all cells were
fed with fresh media as indicated containing
35
S-SO
4
for a second
24-hour period (n¼4). A. Relative ATP content was determined
as described in methods. B. Relative sulphate incorporation was
determined by CPC precipitation as described in methods. Data is
expressed as mean +SEM relative to cells exposed to media with
5.6 mmol/L glucose only for both 24-hour intervals, which is
expressed as 100%. *P50.05 as determined by multiple pair-wise
comparisons. C. The size of the proteoglycans decorin and
biglycan as determined by SDS-PAGE on 4–12% gradient gel
with 3.5% stacking gel, with
14
C-labeled molecular weight
standards. Each lane was loaded with 25 000 dpm. The gel
shown is representative of four separate experiments.
Figure 5. Glucosamine decreases [
3
H]- 2-DG uptake and glucose
consumption. A. Confluent VSMC were exposed to media
containing 5.6 mmol/L glucose with the indicated concentrations
of glucosamine (n¼3). After 24 hours in the indicated conditions
cells were exposed to 0.2 mCi of [
3
H]- 2-DG for 15 minutes and
[
3
H]- 2-DG uptake was determined as described in the Methods
section. Values are expressed relative to total cell protein as
mean +SEM. *P50.01 as determined by multiple pair-wise
comparisons. B. Confluent VSMC were exposed to media
containing 5.6 mmol/L glucose with the indicated concentrations
of glucosamine for 24 hours (n¼3). Media glucose content was
determined after 24 hours in the indicated conditions, and was
subtracted from baseline glucose content (5.6 mmol/L or
25 mmol/L). Values are expressed as mean +SEM. *P50.05 as
determined by multiple pair-wise comparisons.
124 P. J. Little et al.
which effects of glucosamine may be due to ATP
depletion.
There are several reports in support of our data
showing that the effects of glucosamine to decrease
proteoglycan sulphate incorporation and size are due
to ATP depletion. Mannose supplementation of the
culture medium of 13-day-old metanephric kidney
explants (Liu et al., 1992) or rat kidneys (Kanwar
et al., 1992) decreased intracellular ATP levels and
caused decreased synthesis of proteoglycans, but not
other matrix molecules. This effect was partly
reversed by ATP supplementation (Liu et al., 1992).
ATP depletion of chondrocytes has also been shown
to decrease proteoglycan synthesis (Kim et al., 1980;
Baker et al., 1989). Conversely a single ATP bolus to
chondrocytes in culture increased subsequent pro-
teoglycan synthesis (Croucher et al., 2000), an effect
we were not able to demonstrate (data not shown).
The mechanisms by which ATP depletion leads to
modified proteoglycan synthesis are not clear. Proteo-
glycan and GAG synthesis are complex, involve
multiple enzymes, and are not fully understood.
Although many enzymes associated with GAG synth-
esis have been cloned (Sugahara et al., 2000; Habuchi
et al., 2006) the processes by which GAGs are
elongated is not known and therefore it is only possible
to speculate on the mechanism through which cellular
ATP depletion leads to inhibition of GAG chain
synthesis. The processes of uptake of monosaccharide
precursors into the Golgi apparatus may require energy
in the form of ATP and thus relative depletion of
synthetic precursors is one possibility (Toma et al.,
1996). The initiation of GAG synthesis for all
proteoglycans appears to occur via a common linkage
region in which there is sequential addition of Xyl-Gal-
Gal-GluA to a specific serine residue in the core
protein (Lindahl et al., 1966). ATP has been shown to
enhance the activity of galactosyltransferase-I, which
transfers the first galactose to the xylosyl serine in the
synthesis of the linkage region (Higuchi et al.,2001).
Thus, we could also speculate that decreased ATP
availability may limit the activity of galactosyltransfer-
ase-I, and/or other GAG synthetic enzymes, leading to
shorter GAG chains with decreased sulphate incor-
poration. Further experiments will be needed to test
this hypothesis.
In summary, the effect of glucosamine to inhibit the
synthesis of GAG chains on proteoglycans secreted by
VSMCs is due, at least in part, to the action of
glucosamine to deplete cells of ATP. Although in vivo
levels of glucosamine, even after dietary supplemen-
tation, are expected to be below the levels that might
lead to cellular ATP depletion the actions may relate
to flux through metabolic pathways rather than
absolute concentrations and the potential effects of
glucosamine on proteoglycan metabolism in vivo
should be carefully considered. Furthermore, inves-
tigations of the effect of the hexosamine pathway and
the complications of diabetes (Cheng et al., 2006)
should take consideration of the effect of glucosamine
to deplete cells of ATP and its effects which may not
relate to the deleterious actions of high glucose on
cells.
Acknowledgements
This work was supported in part by grants AT00555
and DK35816 (LT) and from the National Health
and Medical Research Council of Australia (268928)
(PJL). We gratefully acknowledge support by the
University of Kentucky Hospital under the Physician
Scientist Program.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
References
Anello M, Spampinato D, Piro S, Purrello F, Rabuazzo AM. 2004.
Glucosamine-induced alterations of mitochondrial function in
pancreatic beta-cells: possible role of protein glycosylation. Am
J Physiol Endocrinol Metab 287:E602–8.
Baker MS, Feigan J, Lowther DA. 1989. The mechanism of
chondrocyte hydrogen peroxide damage. Depletion of intra-
cellular ATP due to suppression of glycolysis caused by
oxidation of glyceraldehyde-3-phosphate dehydrogenase.
J Rheumatol 16:7–14.
Ballinger ML, Nigro J, Frontanilla KV, Dart AM, Little PJ. 2004.
Regulation of glycosaminoglycan structure and atherogenesis.
Cell Mol Life Sci 61:1296–306.
Camejo G, Hurt-Camejo E, Olsson U, Bondjers G. 1993.
Proteoglycans and lipoproteins in atherosclerosis. Curr Opin
Lipidol 4:385–91.
Chang SH, Garcia J, Melendez JA, Kilberg MS, Agarwal A. 2003.
Haem oxygenase 1 gene induction by glucose deprivation is
mediated by reactive oxygen species via the mitochondrial
electron-transport chain. Biochem J 371:877–85.
Cheng DW, Jiang Y, Shalev A, Kowluru R, Crook ED, Singh LP.
2006. An analysis of high glucose and glucosamine-induced
gene expression and oxidative stress in renal mesangial cells.
Arch Physiol Biochem 112:189–218.
Croucher LJ, Crawford A, Hatton PV, Russell RG, Buttle DJ.
2000. Extracellular ATP and UTP stimulate cartilage proteo-
glycan and collagen accumulation in bovine articular chon-
drocyte pellet cultures. Biochim Biophys Acta 1502:297–306.
Habuchi H, Habuchi O, Uchimura K, Kimata K, Muramatsu T.
2006. Determination of substrate specificity of sulfotransferases
and glycosyltransferases (proteoglycans). Methods Enzymol
416:225–43.
Han DH, Chen MM, Holloszy JO. 2003. Glucosamine and
glucose induce insulin resistance by different mechanisms in rat
skeletal muscle. Am J Physiol Endocrinol Metab 285:E1267–
72.
Harpur RP, Quastel JH. 1949. Phosphorylation of d-glucosamine
by brain extracts. Nature 164:693.
Hascall VC, Heinegard DK, Wight TN. 1991. Proteoglycans:
Metabolism and Pathology. In: Hay ED, editor, Cell biology of
extracellular matrix. New York: Plenum Press, pp 149–75.
Higuchi T, Tamura S, Tanaka K, Takagaki K, Saito Y, Endo M.
2001. Effects of ATP on regulation of galactosyltransferase–I
activity responsible for synthesis of the linkage region between
the core protein and glycosaminoglycan chains of proteogly-
cans. Biochem Cell Biol 79:159–64.
Hresko RC, Heimberg H, Chi MM, Mueckler M. 1998.
Glucosamine–induced insulin resistance in 3T3-L1 adipocytes
is caused by depletion of intracellular ATP. J Biol Chem
273:20658–68.
ATP depletion inhibits glycosaminoglycan synthesis 125
Kang J, Heart E, Sung CK. 2001. Effects of cellular ATP depletion
on glucose transport and insulin signaling in 3T3-L1 adipo-
cytes. Am J Physiol Endocrinol Metab 280:E428–35.
Kanwar Y, Yoshinaga Y, Liu Z, Wallner E, Carone F. 1992.
Biosynthetic regulation of proteoglycans by aldohexoses and
ATP. Proc Natl Acad Sci USA 89:8621–5.
Kim JJ, Conrad HE. 1980. Secretion of chondroitin SO4 by
monolayer cultures of chick embryo chondrocytes. J Biol Chem
255:1586–97.
King GL, Brownlee M. 1996. The cellular and molecular
mechanisms of diabetic complications. Endocrinology and
Metabolism Clinics of North America 25:255–70.
Kitagawa H, Izumikawa T, Uyama T, Sugahara K. 2003.
Molecular cloning of a chondroitin polymerizing factor that
cooperates with chondroitin synthase for chondroitin polymer-
ization. J Biol Chem 278:23666–71.
Lindahl U, Roden L. 1966. The chondroitin 4-sulphate-protein
linkage. J Biol Chem 241:2113–19.
Little PJ, Tannock L, Olin KL, Chait A, Wight TN. 2002.
Proteoglycans synthesized by arterial smooth muscle cells in
the presence of transforming growth factor-beta1 exhibit
increased binding to LDLs. Arterioscler Thromb Vasc Biol
22:55–60.
Little PJ, Weissberg PL, Cragoe Jr EJ, Bobik A. 1988. Dependence
of Na þ/Hþantiport activation in cultured rat aortic smooth
muscle on calmodulin, calcium, and ATP. J Biol Chem
263:16780–6.
Liu ZZ, Carone FA, Dalecki TM, Lelongt B, Wallner EI, Kanwar
YS. 1992. Mannose-induced dysmorphogenesis of metaneph-
ric kidney. Role of proteoglycans and adenosine triphosphate. J
Clin Invest 90:1205–18.
Maley F, Lardy HA. 1955. Synthesis of N-substituted glucosa-
mines and their effect on hexokinase. J Biol Chem 214:765–73.
Marshall S, Nadeau O, Yamasaki K. 2004. Dynamic actions of
glucose and glucosamine on hexosamine biosynthesis in
isolated adipocytes: differential effects on glucosamine 6-
phosphate, UDP-N-acetylglucosamine, and ATP levels. J Biol
Chem 279:35313–9.
Nakashima Y, Fujii H, Sumiyoshi S, Wight TN, Sueishi K. 2007.
Early human atherosclerosis: accumulation of lipid and
proteoglycans in intimal thickenings followed by macrophage
infiltration. Arterioscler Thromb Vasc Biol 27:1159–65.
O’Brien KD, Olin KL, Alpers CE, Chiu W, Ferguson M, Hudkins
K, Wight TN, Chait A. 1998. Comparison of apolipoprotein
and proteoglycan deposits in human coronary atherosclerotic
plaques: colocalization of biglycan with apolipoproteins.
Circulation 98:519–27.
Schonherr E, Jarvelainen HT, Sandell LJ, Wight TN. 1991. Effects
of platelet-derived growth factor and transforming growth
factor–beta 1 on the synthesis of a large versican-like
chondroitin sulphate proteoglycan by arterial smooth muscle
cells. J Biol Chem 266:17640–47.
Skalen K, Gustafsson M, Rydberg EK, Hulten LM, Wiklund O,
Innerarity TL, Boren J. 2002. Subendothelial retention of
atherogenic lipoproteins in early atherosclerosis. Nature
417:750–4.
Sugahara K, Kitagawa H. 2000. Recent advances in the study of
the biosynthesis and functions of sulphated glycosaminogly-
cans. Curr Opin Struct Biol 10:518–27.
Tannock L, Little PJ, Wight TN, Chait A. 2002. Arterial smooth
muscle cell proteoglycans synthesized in the presence of
glucosamine demonstrate reduced binding to LDL. J Lipid
Res 43:149–57.
Toma L, Pinhal MA, Dietrich CP, Nader HB, Hirschberg CB.
1996. Transport of UDP-galactose into the Golgi lumen
regulates the biosynthesis of proteoglycans. J Biol Chem
271:3897–901.
Vemuri S, Marchase RB. 1999. The inhibition of capacitative
calcium entry due to ATP depletion but not due to
glucosamine is reversed by staurosporine. J Biol Chem
274:20165–70.
Wells L, Vosseller K, Hart GW. 2003. A role for N-acetylgluco-
samine as a nutrient sensor and mediator of insulin resistance.
Cell Mol Life Sci 60:222–8.
Wight TN, Heinegard DK, Hascall VC. 1991. Proteoglycans:
Structure and function. In: Hay ED, editor. Cell biology of
extracellular matrix. New York: Plenum Press, pp 45–77.
126 P. J. Little et al.
