1 Division of Nephrology, Department of Medicine, University of Mississippi Medical Center and the 2 G. V. "Sonny" Montgomery Veterans Affairs Medical Center, Jackson, Mississippi 39216-4505
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ABSTRACT |
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Hyperglycemia leads to alterations in mesangial cell function and extracellular matrix (ECM) protein accumulation. These adverse effects of glucose may be mediated by glucose metabolism through the hexosamine biosynthesis pathway (HBP). The HBP converts fructose-6-phosphate to glucosamine-6-phosphate via the rate-limiting enzyme, glutamine:fructose-6-phosphate amidotransferase (GFA). We have investigated the effects of high glucose (HG, 25 mM) and glucosamine (GlcN, 1.5 mM) on the synthesis of the ECM protein laminin in a SV-40-transformed rat kidney mesangial (MES) cell line. The roles of protein kinases C (PKC) and A (PKA) in mediating laminin accumulation were also investigated. Treatment of MES cells with HG or GlcN for 48 h increased laminin levels in cellular extracts more than twofold compared with 5 mM glucose (low glucose; LG). The presence of the GFA inhibitor diazo-oxo-norleucine (DON, 10 µM) blocked HG but not GlcN-induced laminin synthesis. HG resulted in a time-dependent increase in total PKC and PKA activities, 57±11.3 (P < 0.01 vs. LG) and 85±17.4% (P < 0.01 vs. LG), respectively. GlcN had no effect on the total PKC activity; however, both glucose and glucosamine increased membrane-associated PKC activity by twofold compared with LG. GlcN stimulated total PKA activity by 47±8.4% (P < 0.01 vs. LG). Similarly, membrane- associated PKA activity was also increased by HG and GlcN ~1.8 and 1.5-fold, respectively. HG and GlcN increased cellular cAMP levels 2.2- and 3.4-fold, respectively. Pharmacological downregulation of PKC by long-term incubation of MES cells with 0.5 µM phorbol 12-myristate 13-acetate (PMA) or inhibition of PKA activity by 2 µM H-8 blocked the effects of HG and GlcN on laminin synthesis. These results demonstrate that glucose-induced laminin synthesis in MES cells is mediated by flux through the HBP and that this stimulation involves PKC and PKA signaling pathways.
diabetic nephropathy; laminin; hexosamine biosynthesis pathway
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INTRODUCTION |
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DIABETIC NEPHROPATHY IS
ASSOCIATED with the accumulation of extracellular matrix (ECM)
proteins in the glomerulus and is represented morphologically by
thickening and expansion of the glomerular basement membrane and the
mesangium (23, 29). Hyperglycemia is an important
contributor to the development of diabetic nephropathy (10). Several laboratories have shown that elevated levels
of glucose in mesangial (MES) and tubular cells cause significant increases in the synthesis and accumulation of matrix components such
as collagen type IV, laminin, fibronectin, and proteoglycans (9,
11, 26, 31, 45). Numerous autocrine/paracrine growth factors
found in renal tissue, namely, transforming growth factor- (TGF-
), ANG II, and insulin-like growth factor I (IGF-I) have been
implicated in ECM accumulation and the development of diabetic nephropathy (3, 17, 32, 34, 43).
High concentrations of glucose lead to increased expression of TGF-
mRNA and bioactivity in both MES and proximal tubule cells (17,
45) and a neutralizing antibody against TGF-
blocked the
glucose-induced increase in collagen synthesis, suggesting a direct
role for TGF-
in hyperglycemia-induced matrix protein accumulation
(33, 41). High glucose also leads to a sustained (days to
weeks) increase in protein kinase C (PKC) activity (2, 38,
39, 42). Agents that activate PKC, including high glucose, also enhance TGF-
mRNA expression and bioactivity (17, 18, 27,
35). cAMP- dependent pathways have been implicated in these
events as well. High glucose leads to a twofold increase in
intracellular cAMP levels in MES cells, and the addition of the cAMP
analog, 8-bromo-cAMP, results in transcriptional activation of type IV
collagen (44). These findings suggest that hyperglycemia may lead to diabetic nephropathy through TGF-
-induced stimulation of
ECM synthesis, and that both protein kinase A (PKA) and PKC may be
involved in mediating these effects.
Recent studies have demonstrated that some of the effects of high
glucose on cellular metabolism are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is
converted to glucosamine-6-phosphate by the rate-limiting enzyme,
glutamine:fructose-6-phosphate amidotransferase (GFA) (as shown in
Fig. 1, Ref. 24). This pathway normally
accounts for ~2% of the total intracellular glucose flux resulting
in the production of UDP-N-acetyl glucosamine (UDP-GlcNAC) and other nucleotide hexosamines that serve as precursors for glycoproteins and glycolipids. Chronic exposure of rat-1 fibroblasts to
high glucose decreases both basal and insulin-stimulated glycogen synthase activity, and overexpression of GFA in these cells mimics these effects of high glucose (5, 6). Moreover, transgenic mice overexpressing GFA in skeletal muscle and fat are insulin resistant (15). These results provide evidence that the
hexosamine pathway may serve as a cellular glucose sensor.
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The HBP has also been linked to glucose-mediated changes in cellular
growth and growth factor expression. High glucose leads to a doubling
of the level of TGF- mRNA in primary cultures of rat aortic smooth
muscle whereas glucosamine, at a much lower concentration, stimulates
TGF-
mRNA levels by six- to sevenfold (7, 25).
Glucosamine is also more potent than glucose in stimulating the
expression of TGF-
mRNA in cultured renal glomerular and proximal
tubule cells (8, 20). These findings suggest that the HBP
mediates some of the metabolic abnormalities associated with
hyperglycemia and may be important in the pathogenesis of diabetic
nephropathy. The present study was initiated to examine the underlying
mechanism(s) by which high glucose and glucosamine induce matrix
protein synthesis in MES cells (33, 41). We investigated
the effect of long-term (48 h) exposure of cells to high glucose and
glucosamine on laminin synthesis as well as on PKC and PKA activities
in SV-40 transformed rat kidney MES cells. Our results show that both
high glucose and glucosamine increase laminin synthesis in MES cells
and that the increase in laminin synthesis is correlated with increases
in PKC and PKA activities. Pharmacological inhibition of PKC or PKA
activity blocks the high-glucose- and glucosamine-induced increases
in laminin levels.
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MATERIALS AND METHODS |
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Materials.
Protein kinase inhibitors, recombinant human transforming growth
factor-1, and antibodies to laminin B1/B2 chain, PKC type III
catalytic domain, cAMP RII regulatory subunit, and actinin were
purchased from Upstate Biotechnology, Lake Placid, NY. The enhanced
chemiluminescence (ECL) system was obtained from Amersham (Arlington
Heights, IL). DMEM and F-12 nutrient mixture (Ham's) were from GIBCO
(Grand Island, NY). [
-32P]triphosphate and
6-diazo-5-oxo-L-norleucine (DON) and azaserine (AZA) were
from ICN (Costa Mesa, CA). P-81 phosphocellulose filter paper was from
Millipore. PKC peptide substrate (pseudosubstrate) and PKA peptide
substrate (Kemptide) were purchased from Pierce. PKA inhibitor, H-8
dihydrochloride, and anti-rat fibronectin antibodies were purchased
from Calbiochem-Novabiochem (San Diego, CA). Biotrak cAMP assay kit was
purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All other
reagents were obtained from Sigma (St. Louis, MO).
Cell culture.
SV-40-transformed rat kidney mesangial cells (American Type Culture
Collection, Rockville, MD) were cultured in medium containing DMEM and
Ham's F-12 (3:1 ratio) supplemented with 7% fetal calf serum (FCS)
and 0.5 mg/ml gentamicin at 37°C in a humidified chamber with a 5%
CO2-95% air mixture. Cells were routinely passaged at confluence every 4 days by using 10-cm dishes. These cells retain many
of the important morphological and biochemical features of differentiated MES cells in primary cultures. For studies of laminin synthesis, monolayers at 30-40% confluence were incubated in the above medium supplemented with 2.25% FCS and the desired
concentrations of D-glucose (5 mM or 25 mM) or glucosamine
(5 mM glucose plus 1.5 mM glucosamine) for 48 h. For
downregulation of PKC or inhibition of PKA activity, 0.5 µM PMA or 2 µM H-8 was added to the medium 3-4 h before adding high glucose
or glucosamine and they were present throughout the time of culture. To
examine for osmolar effects of high glucose in the media, 25 mM
L-glucose with 3 mM D-glucose was utilized
instead of 25 mM D-glucose. At the end of incubation,
culture dishes were rinsed twice with PBS and harvested in 1 ml PBS by
using a rubber policeman. The cells were centrifuged at 16,000 g for 5 s in a microcentrifuge and resuspended in 200 µl of extraction buffer A (50 mM -glycerophosphate, pH
7.3, 0.1% Tween-20, 1.5 mM EGTA, 1 mM dithiothreitol, 0.2 mM Na
orthovanadate, 1 mM benzamidine, 1 mM NaF, 10 µg/ml aprotonin, 20 µg/ml leupeptide, 2 µg/ml pepstatin, and 0.5 µg/ml microcystin
LR). Cell pellets were immediately frozen in liquid nitrogen and stored
at
80°C until use. Cells were subsequently thawed, sonicated for
20 s and centrifuged at 16,000 g for 10 min at 4°C.
To obtain membrane fractions, cells were extracted in buffer
A without detergent and centrifuged as above. The pellet was
washed and resuspended in buffer A plus 1% (vol/vol) Triton
X-100, sonicated for 20 s, and centrifuged at 16,000 g
for 10 min. The supernatant was collected as the membrane fraction.
Protein concentration in extracts was determined by the Coomassie
Protein Assay Reagent from Pierce by using BSA as the standard.
Electrophoresis and immunoblotting.
The soluble cell lysates (30 µg protein in 20 µl) were mixed with 5 µl of a fivefold concentrated SDS sample buffer and heated for 5 min
at 100°C. The proteins were then subjected to SDS-polyacrylamide gel
electrophoresis by using 5% stacking and 10% resolving gels. Proteins
were electroblotted on to a polyvinyledene difluoride filter (PVDF)
membrane. The filter was blocked with 5% nonfat dry milk in
buffer B containing 10 mM Tris · HCl, pH 7.8, 150 mM
NaCl, and 0.05% Tween-20 for 30 min. The membrane was washed twice
with buffer B and incubated overnight with appropriate
antibodies at 4°C with continuous shaking. Antibodies in buffer
B plus 5% dry milk were used at a 1:3,000 dilution for
anti-laminin, 1 µg/ml for anti-PKC and 0.5 µg/ml for anti-
actinin. After incubation with the primary antibodies, the PVDF
membrane was rinsed and washed thrice (10 min each) with water or
buffer B and then incubated for 1.5 h at room
temperature with horseradish peroxidase-conjugated anti-rabbit [for
laminin and
-actinin or anti-mouse (for PKC)] IgG (at a 1:3,000
dilution) in buffer B plus dry milk. After extensive washing
(15 min × 1, 5 min × 2), immunoreactive bands on the
membrane were detected by ECL. For quantitative studies, the
intensities of the bands were measured by a Bio-Rad GS-700 imaging densitometer.
Determination of PKC activity.
PKC peptide substrate (pseudosubstrate), RFARKGSLRQKNV, was
used to measure PKC activity in MES cell extracts. The reaction was
carried out in 30 µl containing 20 mM Tris · HCl, pH 7.5, 10 mM Mg-acetate, 0.9 mM CaCl2, 0.4 mM EGTA, 30 mM
-mercaptoethanol, 25 µg/ml micellar phosphatidylserine, 0.4 µM
PKA inhibitor peptide (PKI), 4 µM compound R24571 (an inhibitor of
Ca2+/calmodulin-dependent protein kinases), 100 µM
pseudosubstrate, 5 µg protein of cell extract, and 250 µM
[
-32P]ATP (800-1,000
counts · min
1 · pmol
1).
After 15 min at 30°C, 25 µl of the reaction mixture were spotted on
P-81 phosphocellulose filters. The filters were washed 4 times (5 min
each wash) with 0.5% (wt/vol) phosphoric acid, and 32P
incorporated into peptides was determined by counting radioactivity in
a liquid-based scintillation counter. The amount of radioactivity associated with cell extracts in the absence of pseudosubstrate was
subtracted to obtain PKC activity. 32P incorporated into
PKC pseudosubstrate without adding cell extracts was negligible. The
protocol for determining the membrane fraction PKC activity is
identical to that of the total PKC activity described above.
PKA activity assay and cAMP determination.
Phosphorylation of PKA peptide substrate (Kemptide),
LRRASLG was carried out in a 30-µl reaction containing 20 mM Tris · HCl, pH 7.5, 100 mM KCl, 1 mM DTT, 15 mM Mg-acetate,
4 µM PKC inhibitor peptide, 4 µM compound R24571, 250 µM
Kemptide, 250 µM [-32P]ATP, and 5-µg cell extract.
After incubation at 30°C for 15 min, the amount of 32P
incorporated into Kemptide was determined by binding to a P-81 filter
as described above for PKC. The addition of 0.5 µM PKA inhibitor,
PKI, to the reaction mixture completely blocked the phosphorylation of
Kemptide, indicating a specific phosphorylation of the peptides by PKA.
The activity of PKA in the membrane fraction was also determined by the
same reaction condition described above. Total cellular cAMP level was
determined after cells were exposed to low glucose, high glucose, or
glucosamine by using the cAMP [125I] scintillation
proximity assay (SPA) kit from Amersham according to the
manufacturer's instructions.
Statistical analysis. Results are expressed as means ± SE of the indicated number of experiments. Student's t-test was used to compare differences between cultures. A P value of <0.05 was considered statistically significant.
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RESULTS |
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Glucose and glucosamine increase laminin synthesis.
Laminin is an ECM component found in virtually all basement membranes,
and the expression of renal laminin B2 mRNA and protein has been
reported to be elevated in diabetic nephropathy (1, 11).
To determine whether cellular laminin expression is increased by high
glucose or glucosamine, MES cells were cultured for 48 h in
DMEM/Ham's medium with 2.25% FCS in the presence of low glucose (5 mM), high glucose (25 mM), or glucosamine (5 mM glucose plus 1.5 mM
glucosamine). Immunoblotting showed that the amount of laminin in
cellular extracts was increased 2.0-and 1.7-fold (P < 0.05) by high glucose or glucosamine, respectively, compared with low
glucose (Fig. 2, A and
B). The effects of high glucose and glucosamine on laminin
only began to be observed after 24 h of incubation (data not
shown). Culturing the cells in DON (10 µM), an inhibitor of GFA,
blocked the stimulation of laminin synthesis by high glucose but not
glucosamine (Fig. 2, C and D), consistent with
the involvement of the HBP in hyperglycemia-mediated laminin production
in MES cells. High glucose and glucosamine had no effect on
alpha-actinin, a structural protein (Fig. 2E). The effects of glucose on laminin synthesis were not due to osmolar changes as 25 mM L-glucose had no effect on laminin levels (Fig.
3).
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The effects of glucose and glucosamine on the ECM are not unique to
laminin.
We sought to see whether the effects of the HBP were limited to laminin
or whether it affected other ECM components. Exposure of MES cells to
high glucose and glucosamine increased fibronectin synthesis, another
ECM protein, by 2.4- and 1.9-fold, respectively, (Figs. 4A).
The addition of AZA, another inhibitor of GFA, resulted in a 68%
decrease in the high-glucose-induced increase in fibronectin synthesis.
Again, the GFA inhibitor had no effect on glucosamine-mediated fibronectin synthesis, supporting further the role of the HBP in
mediating the effects of glucose on the ECM (Fig.
4B).
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Effect of high glucose and glucosamine on PKC and PKA activities.
Agents that stimulate PKC and PKA activities have been shown to
increase extracellular matrix protein synthesis in glomerular MES cells
(27, 35, 37, 44). The effects of high glucose and
glucosamine on PKC and PKA activities in MES cells were therefore determined by using specific peptide substrates. Figure
5A shows that high glucose
stimulated total cellular PKC activity by 57.8 ± 11.3 (P < 0.01 vs. low glucose) while glucosamine had no
effect on the total PKC activity. However, in isolated membrane
fractions, both high glucose and glucosamine-stimulated PKC activity by
109.3 ± 25.7% (P < 0.006, n = 5) and 134.9 ± 51.5% (P < 0.03, n = 5), respectively (Fig. 5B). Neither high
glucose nor glucosamine had any influence on total cellular PKC protein
content on Western blots with anti-type III PKC catalytic domain
antibodies that detect the 82-kDa -,
1-,
2-, and
-isoforms
(21, 38) (Fig. 5C). Similarly, glucose and
glucosamine had no effect on PKC protein content in the membrane
fraction (data not shown).
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Hexosamines regulate laminin via PKC and PKA pathways.
We investigated further the roles of PKC or PKA on high glucose and
glucosamine-induced laminin synthesis. We utilized inhibitors of PKC
(long-term exposure of MES to PMA) and PKA (PKA inhibitor, H-8)
activity. As shown in Fig. 8A,
downregulation of PKC by long-term incubation of cells with 0.5 µM
PMA blocked the effect of high glucose on laminin synthesis completely.
PKA inhibition with 2.0 µM H-8 led to a 44% decrease in
glucose-induced laminin synthesis (P < 0.05 compared
with high glucose alone). Both PMA and H-8 also blocked
glucosamine-induced laminin synthesis (1.6 ± 0.1-fold increase by
GlcN alone); 1.1 ± 0.01 by GlcN plus PMA and 1.1 ± 0.1 by
GlcN plus H-8 (P < 0.03) (Fig. 8B).
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DISCUSSION |
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Numerous studies have demonstrated that high glucose levels cause
an increase in the synthesis and accumulation of ECM proteins, such as,
collagen type IV, laminin, and fibronectin in cultured MES and tubule
cells (3, 12, 17, 32, 34). Some of the effects of high
glucose are mediated by TGF- acting in an autocrine/paracrine fashion. TGF-
increases the accumulation of ECM proteins in these cells, and neutralizing antibodies against TGF-
attenuate the stimulation of matrix proteins by high glucose (33, 41).
Glucose is an important regulator of cell growth and metabolism, and it is likely that some of the adverse effects of high glucose are mediated
by normal regulatory pathways. The HBP, which converts fructose-6-phosphate to glucosamine-6-phosphate with glutamine as the
amino donor, has been hypothesized to be a sensor for glucose and
therefore a mediator of glucose regulation in a variety of cell types
(5-8, 15, 24, 25). In kidney MES cells, glucosamine was more potent than glucose in stimulating TGF-
mRNA transcription and bioactivity (8, 20), and the inhibition of GFA
activity by the glutamine analogue AZA or antisense oligonucleotide
against GFA blocked the high glucose-induced expression of TGF-
and
matrix protein synthesis (20). Likewise, hexosamines
regulate TGF-
1 transcription in rat kidney cells (mesangial and
proximal tubule) and rat vascular cells via regulation of the TGF-
1
promoter (8, 33).
We sought to investigate whether the effects of high glucose on ECM are mediated by the HBP. Here, we show that glucosamine mimicks glucose in its ability to increase laminin synthesis. These effects of high glucose and glucosamine required at least 24 h before they were observed. The effects of hexosamines are not specific to laminin alone as both high glucose and glucosamine also increased fibronectin synthesis in MES cells. The role of the HBP in mediating the effects of glucose on the ECM is supported by the ability of two inhibitors of GFA to blunt the high glucose-induced increases in laminin and fibronectin. However, glucosamine-enhanced ECM synthesis is not affected by these GFA inhibitors as glucosamine enters the HBP distal to the rate-imiting enzyme GFA.
These observations support the notion that the HBP mediates the effects of glucose in the mesangium. Kolm-Litty et al. (20) also reported that treatment of primary cultures of porcine glomerular MES cells with 12 mM glucosamine for 48 h leads to a 2.3-fold increase in fibronectin synthesis. In the present study, the effect of glucosamine on laminin and fibronectin was observed at a much lower concentration than glucose (1.5 mM glucosamine vs. 25 mM glucose), and, in fact, in our experimental system glucosamine concentrations greater than 2.5 mM drastically reduced cell growth and viability. The reason for this discrepancy may be the differences in cell types used for the studies, but it is an important consideration given the potential, specific untoward effects of glucosamine in certain cells (14). Nevertheless, the fact that others have seen similar effects in primary cells makes it less likely that the observations reported here are unique to a transformed cell line. In additon, the importance of the HBP on mediating the assembly of the ECM is supported by the demonstration of increased fibronectin secretion in the studies mentioned above (20) and in preliminary data from our laboratory that demonstrates increased secretion of laminin in MES cells cultured in high glucose and glucosamine (not shown).
The underlying mechanism(s) by which high glucose and glucosamine
enhance matrix protein synthesis are not fully understood. PKC is a
candidate mediator for the induction of TGF- synthesis by high
glucose because agents, including high glucose, that increase PKC
activity also increase TGF-
transcription and bioactivity (2,
27, 35, 38, 39, 42). The role of PKC in laminin synthesis is not
entirely clear as recent reports have not supported a role for PKC in
the regulation of the laminin C1 promoter (30). Similarly,
an increase in PKC activity as such may not be required for
TGF-
-mediated matrix protein production (36).
Like their effects on laminin, high glucose and glucosamine increased membrane PKC activity in MES cells. This indicated a possible role for PKC in HBP-mediated ECM regulation. It was confirmed that hexosamines increase laminin via a PKC mechanism as long-term exposure of cells to PMA blocked the glucosamine-induced increase in laminin. Unlike high glucose, we did not see an effect of glucosamine on total PKC activity. However, in membrane fractions, both high glucose- and glucosamine-stimulated PKC activity are about twofold, suggesting a role for the hexosamine pathway in PKC distribution between cytosolic and membrane fractions.
There are at least 11 PKC isoforms and they are categorized into 3 subclasses according to their structure and function. PKC isoforms,
,
1,
2, and
are classified in the conventional group which
are regulated by diacylglycerol, phosphatidylserine, and
Ca2+; novel PKCs (
,
,
and
), which are also
regulated by diacylglycerol and phosphatidylserine; and atypical (
,
, and
), whose regulation has not been clearly established,
although their activity can be stimulated by phosphatidylserine
(12, 28). We did not find any change in protein content of
conventional PKCs on Western blots with anti-type III PKC antibodies
which recognize PKC
,
1,
2, and
. Different PKC isoforms,
however, have different enzymatic properties and their distribution
changes after cell activation. Some isoforms are translocated from the
cytosolic compartment to cellular membranes while others are
translocated into the nucleus where they play a major role in signaling
(12, 19). It is not known which of the isoforms are
activated or translocated by high glucose or glucosamine. Answering
this question and how these PKC isoforms participate in ECM regulation
will be the focus of future experiments.
Hyperglycemia also increases the intracellular concentration of cAMP
(22, 44) and, therefore, may activate the PKA pathway in
MES cells. We observed that high glucose increases cAMP content in MES
cells and glucosamine mimics the effect of high glucose on cAMP levels.
As observed with PKC, hexosamines appear to be involved in PKA
translocation, however, unlike PKC, hexosamines appear to regulate PKA
at the protein level (Fig. 6C). The ability of high glucose
and glucosamine to increase PKA activity and the ability of the PKA
inhibitor, H-8, to decrease their effects on laminin synthesis, suggest
that a PKA-mediated pathway may be important in ECM regulation. This is
supported by the work of other investigators that has demonstrated
regulation of PKA by TGF- in the mesangium (37).
We hypothesize that the HBP acts as a cellular glucose sensor in target
cells. Abnormalities in flux through or regulation of this pathway may
lead to altered cellular responses to glucose. This is supported by the
loss of glucose-induced increases in laminin levels when GFA, the rate
limiting enzyme in the HBP is inhibited by glutamine analogs like DON
or AZA. Further support of this hypothesis is the observed
downregulation of GFA activity (the rate limiting enzyme in this
pathway) by high glucose in MES cells (4). Thus when flux
through the HBP is altered, downstream products of this pathway may
upregulate PKC, PKA, and TGF- bioactivity leading to increased ECM
gene expression (8, 20) and protein synthesis. The
upregulation of these signaling systems in MES cells ultimately leads
to increased ECM levels. The effects of PKC and PKA inhibition on
glucose- or glucosamine-induced laminin synthesis (Fig. 8) strongly
support the involvement of these intracellular signaling pathways in
glucose-mediated ECM synthesis.
How the HBP mediates the ECM and how PKC and PKA are involved in this process are not completely understood. The time course for the effects of hexosamines on PKC, PKA, and ECM components (>24 h) is consistent with transcriptional regulation of some regulatory gene(s). An interesting hypothesis for how the HBP has its effects is through O-linked protein glycosylation (14, 24). The substrate for this posttranslational modification of proteins is UDP-GlcNAC (Fig. 1) a downstream metabolite of the HBP (24). Among the proteins that are O-glycosylated are transcription factors and this process is mediated by hexosamine metabolism (13). UDP-GlcNAC is increased in our cells when cultured in high glucose or GlcN (not shown). We propose that increased flux through the HBP (as seen with hyperglycemia) leads to increased UDP-GlcNAC levels and altered regulation of genes that mediate synthesis of the ECM. Included among these are genes encoding proteins that regulate PKC and PKA. This hypothesis is supported by the fact that PKA protein levels did change with glucose and glucosamine. This ultimately may lead to elevated ECM levels and diabetic gluomerulosclerosis. Ongoing work is focused on uncovering the mechanism by which glucose metabolism through the HBP regulates ECM.
In conclusion, the present study demonstrates that high-glucose-induced laminin synthesis in MES cells is mediated by the HBP. Glucosamine, at a much lower concentration, mimics the effects of high glucose. DON, an inhibitor of GFA, blocks the high glucose-, but not glucosamine-induced increases in laminin synthesis. The data also suggest that both PKC and PKA signaling pathways may participate in high-glucose and glucosamine regulation of matrix protein production and that these proteins are regulated by the HBP. Elucidation of the various signaling pathways mediated by hyperglycemia and excess hexosamines may allow the development of novel means for therapeutic intervention and treatment of patients with diabetic nephropathy.
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ACKNOWLEDGEMENTS |
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Support for this work was from The Robert Wood Johnson Foundation (E. D. Crook) and Kidney Care Foundation. E. D. Crook is also supported by a Career Development Award from the Veterans Administration. Portions of this work were presented at the 1997 American Society of Nephrology Meeting in San Antonio, TX.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. D. Crook, Dept. of Medicine, Div. of Nephrology, 2500 N. State St., Jackson, MS 39216-4505 (E-mail: ecrook{at}medicine.umsmed.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 November 1999; accepted in final form 5 June 2000.
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