Departments of Pathology and Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
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ABSTRACT |
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Troglitazone is a peroxisome proliferator-activated receptor-
agonist that has been shown to halt mesangium expansion in experimental
models of type 2 diabetes mellitus and to act directly on rat mesangial
cells. Because glutamine serves as the precursor for cellular
biosynthetic processes, we asked whether troglitazone would inhibit
mesangial cell glutamine metabolism under these conditions. Confluent
monolayers of rat mesangial cells were incubated in RPMI medium in the
presence of troglitazone or vehicle (DMSO). Troglitazone effected a
dose-dependent reduction in glutamine utilization and in alanine
formation, associated with a decrease in monolayer
collagen-glycosaminoglycan content. Despite the reduced glutamine
uptake, ammonium formation did not decrease, consistent with increased
glutamate flux through the deamination pathway. Assayable activity of
the alanine aminotransferase decreased by 63%, whereas assayable
glutamate dehydrogenase remained unchanged. In control monolayers, the
sum of ammonium plus alanine plus glutamate nitrogen released accounted
for <75% of the glutamine nitrogen uptake. In troglitazone-treated
monolayers, all of the glutamine nitrogen taken up could be accounted
for as ammonium nitrogen released into the medium. These results are
consonant with troglitazone reducing glutamine metabolism and
specifically the transamination pathway in rat mesangial cells
associated with a reduction in collagen-glycosaminoglycan content.
peroxisome proliferator-activated receptor-; alanine
aminotransferase; matrix proteins; intracellular pH
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INTRODUCTION |
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TROGLITAZONE IS A THIAZOLIDINEDIONE that exhibits insulin-sensitizing activity and has been shown to attenuate hyperinsulinemia and hyperglycemia in human subjects with type 2 diabetes mellitus (5). In addition to its insulin-sensitizing activity, troglitazone halts mesangium expansion and reduces proteinuria in insulin-resistant diabetic animals (10, 14), as well as reducing production of extracellular matrix in isolated mesangial cells (24). These findings point to troglitazone actions other than its insulin-sensitizing activity in ameliorating the expansion of extracellular matrix occurring in diabetic states.
We chose to study the action of troglitazone on mesangial cell glutamine metabolism because of troglitazone's effect of inhibiting matrix expansion in vivo (10, 12, 14) and in vitro (24) and because glutamine supports protein synthesis (9, 20). However, it is not known whether mesangial cells utilize glutamine, because arginine is present as the other major nitrogenous precursor in the standard RPMI medium (16). Thus there are two potential substrates available for protein synthesis in vitro, and it is not known whether troglitazone inhibits utilization of these precursors. Therefore, our purpose was to determine whether troglitazone inhibits amino acid utilization under in vitro conditions in which matrix protein production is limited (24), and if so, whether this effect is limited to glutamine and to a specific pathway.
The major fuels both in vivo for the intact functioning kidneys
(21) and for in vitro culture conditions (22)
are glutamine and glucose, and the putative metabolic pathways involved
in their utilization by mesangial cells in culture are shown in Fig.
1. Although troglitazone has been shown
to directly enhance glucose uptake and accelerate glycolysis
(7), no previous information is available concerning an
effect of troglitazone on amino acid metabolism. Thus monitoring
glucose uptake and lactate production provides a positive control for
troglitazone's only known metabolic action. Troglitazone-enhanced
glucose uptake in muscle (7) is coupled to the formation
of lactate with the possibility of 2 mol lactate/mol glucose utilized.
Whether and how glutamine is metabolized by mesangial cells in culture
are not known; however, these cells do not express the
phosphate-independent glutaminase found in most
epithelial cells so that glutamine hydrolyzed to glutamate and
ammonium is by the intracellular glutaminase (Fig. 1, reaction
1). The pivotal intermediate is glutamate, because it can either
be transaminated (Fig. 1, reaction 2) by alanine aminotransferase (ALT), present in both the cytosol and mitochondria (13), or deaminated (Fig. 1, reaction 3) by the
mitochondrial glutamate dehydrogenase (GDH) (17, 27).
Nevertheless, glutamate metabolized by both pathways yields the same
carbon product, -ketoglutarate (AKG2
), which can be
oxidized in the tricarboxylic acid cycle via pyruvate formation
(17, 27). Note that protein synthesis can also be dependent on glutamine utilized as an oxidative fuel (9).
The nitrogen products are very different according to the respective pathway utilized: deamination (Fig. 1, reaction 3) yields
ammonium released into the medium, whereas transamination (Fig. 1,
reaction 2) produces alanine (and other amino acids,
depending on the dicarboxylic acid-nitrogen acceptor), which may
undergo further transamination reactions and serve as precursors for
matrix protein production. Consequently, glutamine may support
mesangial cell protein synthesis either as an energy fuel or nitrogen
source or both.
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To determine whether troglitazone inhibits glutamine utilization and to assess the site(s) of inhibition, confluent rat mesangial monolayers were incubated with troglitazone, and glutamine uptake as well as the nitrogenous products formed was determined. The inhibitory effect of troglitazone on glutamine metabolism was then related to the monolayer total protein and collagen-glycosaminoglycan content. The results to follow demonstrate that troglitazone reduces glutamine metabolism by virtually eliminating the transamination pathway associated with a reduction in mesangial cell collagen-glycosaminoglycan content.
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MATERIALS AND METHODS |
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Cell culture. Glomerular mesangial cells were cultured as previously described (11, 24). Briefly, glomeruli were isolated by differential sieving from kidneys obtained from male Sprague-Dawley rats weighing 300 g. Isolated glomeruli were then cultured in RPMI 1640 medium (BioWhitaker, Walkersville, MD) containing 17% fetal calf serum and antibiotics. Mesangial cells were identified by their stellate shape and their ability to form hillocks in culture. The cells were seeded in either 24- or 12-well plates and used for experiment after gaining confluence. After confluence, the cells were exposed to serum-free RPMI medium (16) containing 5 mM glucose and either 5, 10, or 20 µM troglitazone (gift of Dr. Hardwick Johnson, Parke-Davis, Ann Arbor, MI) dissolved in DMSO or DMSO alone. The medium was then harvested and the cells washed with ice-cold PBS (3×) and lysed in 50 mM Tris buffer containing 0.1% Triton X-100 and the protease inhibitors (in mM) 0.2 phenylmethylsulfonyl fluoride, 5 benzamidine, and 10 EDTA, pH 7.2. Aliquots of the medium were processed for amino acid analysis by HPLC (30) or used directly for enzymatic analysis of glucose and lactate or microdiffusion analysis of ammonia. Cell lysates were used for ALT, GDH, total protein, and collagen assays.
Amino acid analysis.
Medium samples were promptly treated with an equal volume of ice-cold
5% trichloroacetic acid, left on ice for 10 min, and then centrifuged
at 10,000 g for 10 min. Aliquots of the protein-cleared supernatant containing free amino acids were then treated with O-phthalaldehyde (FLUKA, Buchs, Switzerland) for precolumn
derivatization and injected onto a C18 4.6 × 250 mm
column (Microsorb, Varian, Walnut Creek, CA) for separation of the
derivatized amino acids. The column effluent was passed through a
fluorescence detector, with peaks for the major amino acids at
characteristic retention times as shown in Fig.
2. Figure 2A shows the
retention times for standards: aspartate, 7.85; glutamate, 11.2;
serine, 15.7; glutamine, 16.4; homoserine (internal standard), 17.8;
arginine, 18.3; and alanine, 20.5 min (Asp and Arg at 0.5 mM; all
others 1 mM). Figure 2B shows the amino acid profile of the
serum-free RPMI 1640 medium incubated for 72 h in the absence of
cells. Note the absence of an alanine peak at 20.5 min in the
serum-free RPMI 1640 medium. Figure 2C shows the effect of
incubating mesangial cells for 72 h on the amino acid profiles.
Figure 2D shows the effect of incubating mesangial cells
plus 10 µM troglitazone on the same amino acid profile; note the
ability to monitor the major amino acids involved in nitrogen balance
as well as to detect the diminished alanine peak. The medium
concentration of the major amino acids was obtained from the peak areas
in B, C, and D divided by the peak areas shown in
A for their respective standards. Utilization or production
rates for the respective amino acids were obtained from the
concentration differences observed between B and either C (control) or D (troglitazone treated) times the
medium volume, 1-2 ml. These rates were then expressed as
milligrams of total protein.
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Total protein, collagen, and glycosaminoglycan assays. Total protein in the monolayers was determined in triplicate on the cell lysates by using the dye-binding assay with albumin as the standard (1). Total monolayer collagen content was determined as the total soluble collagen by using the Sircol Collagen Assay kit (Biocolor, Belfast, Northern Ireland). Aliquots of the cell lysates were added to 1 ml of Sircol dye reagent that binds to collagen, followed by 30 min of mixing. After centrifugation at 10,000 g for 10 min, the supernatant was carefully decanted, and 1 ml of 0.5 M NaOH was added. Samples and collagen standards were then read at 540 nm on a spectrophotometer. Collagen concentration in the lysate buffer was then read from a standard curve with the use of acid-soluble type 1 collagen. Collagens contain ~14% hydroxyproline, and collagen concentration obtained using Sirus Red binding correlates well with the hydroxyproline content (Sircol Collagen Assay, Biocolor). The sulfated proteoglycan and glycosaminoglycan content of mesangial cell monolayers was determined in the aforementioned cell lysates with a quantitative dye-binding assay (Blyscan Proteoglycan & Glycosaminoglycan Assay, Biocolor). The assay uses 1,9-dimethylmethylene blue as a dye label, with sample concentrations determined using a calibration curve prepared with glycosaminoglycan standards (0-5.0 µg).
Ammonium determination. Ammonium concentration was determined by the microdiffusion method (30), and formation rate was determined as described by subtracting the medium blank and expressing it as milligrams of protein.
Glucose and lactate determination. Glucose concentration was measured enzymatically with hexokinase (Sigma, St. Louis, MO) and glucose-6-phosphate dehydrogenase (Sigma), the latter catalyzed reaction converting glucose 6-phosphate and NAD to 6-phosphogluconate and NADH; coupled NAD conversion to NADH was monitored at 340 nm. Lactate concentration was determined enzymatically using lactate dehydrogenase (Sigma) coupled to the reduction of NAD to NADH monitored as described. The assay conditions included a hydrazine trap for pyruvate to force the reaction to completion. Utilization of glucose and production of lactate were calculated as for the amino acids and are expressed as milligrams of protein.
Enzymatic assays.
Both of the enzymes catalyzing the conversion of glutamate to AKG, Fig.
1, reactions 2 and 3, were assayed
using the cell lysate and carried out under optimal conditions as
follows. ALT activity was assayed in the reverse direction to that
shown in Fig. 1, reaction 2, by use of 1 M
alanine and 0.3 M AKG to produce glutamate and pyruvate
(32); conversion of pyruvate to lactate was then coupled
to NADH oxidation in the presence of lactate dehydrogenase, with the
rate of decrease in absorbency at 340 nm being directly proportional to
the amount of cell lysate buffer protein added. GDH activity was
assayed in the reverse direction to that shown in Fig. 1,
reaction 3, using 3.3 M NH
Statistical analysis. Differences between control and troglitazone-treated monolayers were analyzed using either Student's t-test or ANOVA and a corrected t-test (Bonferroni) for multiple doses with differences considered significant at P < 0.05.
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RESULTS |
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As shown in Fig. 2C, glutamine, and not arginine, was
the major nitrogen source for mesangial cell protein synthesis, and its
uptake was reduced in response to troglitazone (Fig. 2, C vs. D). Therefore, we focused on glutamine uptake and
specifically the products of the pathways depicted in Fig. 1. The
response of glutamine uptake and alanine and ammonium formation to 5, 10, and 20 µM of troglitazone for 24 h is shown in Fig.
3, A, B, and C,
respectively. Glutamine uptake (Fig. 3A) was decreased at 10 (P < 0.01) and 20 µM (P < 0.01)
troglitazone by 32 and 37%, respectively (961 ± 67 and 884 ± 68, respectively, vs. 1,406 ± 69 nmol/mg protein for control),
whereas the 20% decrease at 5 µM troglitazone did not achieve
statistical significance (1,125 ± 78, P < 0.10).
In contrast, alanine formation (Fig. 3B) was reduced by 72, 84, and 95% (from 868 ± 110 to 240 ± 35, 135 ± 11, and 40 ± 12 nmol/mg protein) at 5, 10, and 20 µM troglitazone,
respectively (all P < 0.01, ANOVA and corrected
t-test). Ammonium production (Fig. 3C) increased
1.6-fold at 5 µM troglitazone (2,036 ± 242 vs. 1,262 ± 113 nmol/mg protein, P < 0.05 vs. control), followed
by a decline to values not different from the controls at 10 and 20 µM despite the reduced glutamine utilization. These results show that
troglitazone markedly inhibits alanine formation in a dose-dependent
manner, with a reduction in overall glutamine utilized (significant at the higher doses, 10 and 20 µM, of troglitazone).
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Figure 4 shows the formation of alanine
by control monolayers and by monolayers exposed to 10 µM troglitazone
over a 72-h time course. At 12 h, troglitazone decreases the
formation of alanine by 73% (6.8 ± 0.2 vs. 25 ± 2 nmol · h1 · mg
1 for
control, P < 0.001), whereas glutamine uptake was
unchanged (81 ± 12 vs. 75 ± 10 nmol · h
1 · mg
1,
respectively). After an additional 12 h, the alanine formation decreases to only 9% of the control monolayer (28 ± 4 vs.
2.4 ± 2 nmol · h
1 · mg
1,
P < 0.004). At 48 and 72 h, troglitazone still
suppresses alanine formation to a rate that is less than 20% of the
control monolayer alanine formation rate.
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Figure 5 shows the total protein and
collagen content of mesangial monolayers incubated for 48 h with
vehicle or 5, 10, and 20 µM troglitazone. Total protein content
decreased at 20 µM troglitazone by 38%, but the total protein
content in monolayers treated with 5 and 10 µM was not significantly
reduced. In contrast to the total protein, collagen content
significantly decreased (29%, P < 0.05; 48%,
P < 0.01; 52%, P < 0.01) at 5, 10, and 20 µM troglitazone, respectively. These results show that
troglitazone markedly reduces the collagen content of mesangial cells
and can do so at a concentration of troglitazone (5 µM) that inhibits
the transamination but has little inhibitory effect on glutamine
utilization.
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The effects of incubating mesangial cells with 10 µM troglitazone on
glutamine and glucose uptake are shown in Fig.
6. Mesangial cells readily take up
glucose and released lactate, whereas troglitazone increased both
glucose uptake (10,684 ± 198 vs. control, 8,671 ± 50 nmol/mg, P < 0.01) and lactate release (9,851 ± 1,150 vs. control, 6,344 ± 413 nmol/mg, P < 0.05). This confirms that troglitazone's enhancement of glucose
utilization observed in other cells (7) can also be
demonstrated in cultured mesangial cells. Noteworthy, troglitazone
increased lactate formation relatively more than the glucose uptake (55 vs. 23%), consistent with a shift to anaerobic glycolysis as
previously noted (7). In contrast to the increase in
glucose uptake, troglitazone decreased glutamine uptake by 30%
(1,525 ± 231 vs. 2,176 ± 376 nmol/mg, P < 0.01) and glutamate release by 41% (245 ± 35 vs. 413 ± 60 nmol/mg, P < 0.005).
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Table 1 presents the balance between
nitrogen taken up as glutamine's amino and amide nitrogen
(glutamine × 2) and the nitrogen released as the sum of ammonium,
glutamate, and alanine. In the absence of troglitazone, only 73% of
the glutamine nitrogen removed could be accounted for as the sum of
ammonium, alanine, and glutamate nitrogen (3,192/4,350 nmol/mg × 100). Therefore, in the control monolayers there is a large positive
nitrogen balance. In contrast, with 10 µM troglitazone, all of the
glutamine nitrogen could be accounted for, predominantly as ammonium
(3,106/3,050 nmol/mg). As shown in Fig. 2, C and
D, serine was taken up in the control monolayers (383 ± 20 nmol/mg), and this uptake reversed to a small release with
troglitazone (63 ± 18 nmol/m, P < 0.0001 vs.
control uptake). After 72 h, the total protein content remained
unchanged (1,229 ± 32 and 1,132 ± 42 mg/monolayer), whereas
collagen content was decreased by 40% (40 ± 2 to 24 ± 2 µg/monolayer, P < 0.001), and glycosaminoglycan
content decreased by 38% (5.8 ± 0.3 to 3.6 ± 0.3 µg/monolayer, P < 0.01).
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The decrease in alanine production shown above was associated with a
63% reduction (6 ± 1 vs. 16 ± 2 U/mg, P < 0.05) in the assayable alanine aminotransferase activity, as shown in
Fig. 7. Addition of troglitazone
(10-100 µM) directly to the assay medium did not inhibit ALT
activity of the control homogenates. On the other hand, cellular
alanine content decreased (P < 0.01) in
troglitazone-treated monolayers from 232 ± 20 nmol/mg protein in
control to 72 ± 6 nmol/mg protein at 10 µM troglitazone
(P < 0.01). Although ammonium formation increased by
47% (3,106 ± 336 vs. 2,115 ± 402 nmol/mg,
P < 0.05), the assayable glutamate dehydrogenase
activity remained unchanged (13 ± 1 vs. 14 ± 2 U/mg; Fig.
7).
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DISCUSSION |
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Thiazolidinediones are agonists (2) for the nuclear
receptor peroxisome proliferator-activated receptor- (PPAR
),
which plays an important regulatory role in the metabolism of lipids and glucose (5, 6). An effect of troglitazone on glutamine metabolism is novel and, if through PPAR
, would provide the first evidence that this pluripotential receptor regulates glutamine metabolism. Because troglitazone has been shown to limit extracellular matrix formation by mesangial cells in vivo in diabetic (10, 14) and nondiabetic animal models (12), and because
matrix protein synthesis is, in part, dependent on glutamine (3,
28, 29), we monitored the effect on both glutamine metabolism
and extracellular matrix production as reflected in the collagen and glycosaminoglycan content. Our results suggest a relationship between
glutamine metabolism and mesangial cell matrix formation, specifically through troglitazone inhibition of the transamination pathway(s). We will discuss first the evidence for the inhibition of
glutamine transamination by troglitazone and then the consequence of
this effect for overall glutamine metabolism; finally, we will relate
this, as far as possible, to the decrease in extracellular matrix protein.
These results demonstrate a novel action of troglitazone of inhibiting glutamine-dependent alanine formation. The evidence for this is that troglitazone decreases alanine formation in a dose-dependent manner (Fig. 3B) associated with a decreased glutamine utilization (Fig. 3A; Table 1). We know that this represents a reduction in glutamate transamination, because parallel studies in monolayers incubated with [2-15N]glutamine show that troglitazone inhibits 15N alanine production (31). We know that this effect of reducing glutamate transamination is not secondary to the reduction in glutamine uptake, because the inhibition of alanine formation quantitatively exceeded the decrease in glutamine uptake (Fig. 3, A and B); furthermore, the decrease in alanine formation occurring at 12 h (Fig. 4) preceded any decrease in glutamine uptake. These results indicate that the site of inhibition was not primarily at the glutamine uptake or glutaminase step but point to the transamination site. In addition, troglitazone reduced the activity of the enzyme carrying out this reaction, ALT activity (Fig. 7), supporting the flux measurement. Taken together, these findings are supportive of troglitazone's primary site(s) of action being at the transamination pathway.
Because the inhibition of alanine formation exceeded the reduction in glutamine uptake, the question arises as to the fate of glutamate formed by the glutaminase reaction (Fig. 1). Clearly, there was not an increased efflux of glutamate from these cells in the presence of troglitazone, rather just the opposite (Fig. 6). However, there was a large increase in ammonium formation (nearly 800 nmol/mg at 5 µM; Fig. 3C) that approximates the decrease in alanine formation (Fig. 3B). These findings are consistent with an accelerated flux of glutamate through the deamination pathway (18, 27). At higher concentrations, troglitazone inhibited glutamine utilization but not the ammonium formation (Fig. 3C), suggesting that the flux through the GDH pathway was maintained despite the reduced overall glutamine utilization. In support of this, studies with [2-15N]glutamine have shown that troglitazone enhances the flux of glutamate through the GDH pathway, leading to the formation of labeled ammonium (31). However, unlike the inhibition of the alanine formation, assayable GDH activity was not increased (Fig. 7). These results are therefore consistent with troglitazone acting to inhibit the transamination pathway and also accelerating glutamate flux through the deamination pathway (Fig. 1). Whether this reflects multiple sites of troglitazone action or a single action that inhibits flux through the transamination while accelerating flux through the deamination pathway remains to be determined.
Troglitazone's effect of inhibiting glutamine metabolism is novel, although its non-insulin-like enhancement of glucose uptake has been demonstrated in vitro (7). Because glucose utilization may be reciprocally related to glutamine utilization (34), enhanced glucose uptake might primarily inhibit glutamine utilization and secondarily reduce the flux through the transamination pathway. However, as discussed above, alanine formation was decreased prior to and was quantitatively greater than glutamine utilization, so that the effect of glucose uptake would have to be primarily at the transamination site. A mechanism for glucose utilization to specifically inhibit the transamination reaction is not readily apparent. Increased lactic acid formation, if it led to an intracellular acidosis, might account for the increase in glutamate flux through the deamination pathway (18, 27). If the transamination reaction was also inhibited with a cellular acidosis, then much of what is observed could be explained by a decrease in intracellular pH.
Other mechanisms to explain the reduced transamination flux could be
considered. Because ALT operates at near equilibrium in cells
(13), a rise in intracellular alanine might occur, and
this would drive the reaction in reverse. However, cellular alanine
concentration actually decreased with troglitazone and therefore favors
the transamination of glutamate, pointing to a reduction in the
activity of the enzyme. Note that this is not a direct effect, because
adding up to 100 µM troglitazone to the assay medium did not inhibit
the activity. On the other hand, assayable ALT activity is reduced
after exposure to troglitazone, consistent with a reduction in the
amount of ALT protein. One possible mechanism for troglitazone to
downregulate ALT is through PPAR, suggesting that
troglitazone may act as a partial agonist (2), possibly by
competing for this receptor's ligand-binding site with a true agonist
(19). Note that serine/pyruvate/alanine glyoxalate
aminotransferase plays an important role in serine metabolism
(33), and although we did not assay for this activity, troglitazone virtually eliminated serine uptake. The observation that
aminotransferase activities may be upregulated by PPAR
activation raises the possibility that transamination reactions are important pathways through which troglitazone exerts its antidiabetigenic action (as antagonists, with reduced gluconeogenesis) and, considering the importance of hepatic alanine uptake, possibly contributing to
hepatic injury.
The increase in ammoniagenesis promoted by troglitazone was not associated with an increase in assayable GDH activity, suggesting that cell pH may be decreased (31). One potential pathway for troglitazone to reduce the intracellular pH would be to decrease protein kinase C activity (10), which in turn limits the activity of sodium/hydrogen ion exchange (31). Another mechanism for inducing an intracellular acidosis might be through an increased acid load, resulting from enhanced lactic acid production (Fig. 6). Further studies designed to test these putative mechanism(s) involved in regulating both alanine and ammonium formed are necessary.
Glutamine has been reported to influence the synthesis of matrix
protein through both the amino and amide nitrogens (3, 20, 28,
29) and to support the energy demands for protein synthesis as
an oxidizable fuel (9). Thus glutamine potentially plays
multiple roles as a provider of precursor amino acids through transamination reactions as well as by providing the amide nitrogen for
glycosaminoglycan synthesis and more generally by providing energy for
protein synthesis. Our observations show that matrix protein content is
reduced by as much as 30% at 5 µM troglitazone concentration without
a corresponding reduction in either total protein content (Fig. 4) or
glutamine uptake (Fig. 3A). These observations argue against
a role for glutamine solely as a source of energy for overall protein
synthesis. Because both collagen and sulfated proteoglycans plus
glycosaminoglycans fall almost 40% with >80% inhibition of the
transamination pathway, this suggests that troglitazone inhibition of
transamination may be coupled in some manner to the reduction in matrix
protein formation. However, we have not measured collagen synthesis, so
that the decrease in total collagen might reflect either a decreased
synthesis or an increased catabolism, or both. Under hyperglycemic and
even normoglycemic conditions, collagen synthesis is associated with increased glucose transporter (GLUT-1) activity (4, 8), and agents that enhance glucose uptake also increase the synthesis of
extracellular matrix components (4). In this regard,
increased glucose uptake coupled to the hexosamine pathway has been
shown to stimulate transforming growth factor- (TGF-
), production (26), and although we did not assay for TGF-
, others
have shown that production of this prosclerotic cytokine is
downregulated by troglitazone in vivo (10, 12). TGF-
may upregulate glutaminase activity and transamination reactions and
thereby provide precursors for subsequent matrix protein synthesis,
e.g., proline, glutamate, and aspartate. Regardless of the mechanism, a
reduction in glycosaminoglycan content of mesangial cells may, in turn,
limit the availability and activation of associated positively charged
chemokines (15, 26) that modulate numerous cell functions
through effects on matrix proteins. Thus troglitazone actions on these
cellular functions may be, in part, through effects on glutamine
metabolism and secondarily may contribute to the modulation of
pathophysiological processes.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the American Diabetes Association and National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-48055 to K. McCarthy) and the Southern Arizona Foundation (T. Welbourne), as well as an American Heart Association Fellowship (R. Routh).
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Welbourne, Dept. of Molecular and Cellular Physiology, LSUHSC, Shreveport, LA 71130 (E-mail: twelbo{at}lsuhsc.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 6 June 2001; accepted in final form 28 August 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bradford, MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
2.
Camp, HS,
Ou L,
Wise SC,
Hong YH,
Frankowski CL,
Shen X-Q,
Vanbogelen R,
and
Leff T.
Differential activation of peroxisome proliferator-activated receptor- by troglitazone and rosiglitazone.
Diabetes
49:
539-547,
2000[Abstract].
3.
Clark, CC,
Richards CF,
Pacifici M,
and
Iozzo RV.
The effects of 6-diazo-5-oxo-L-norleucine, a glutamine analog, on the structure of the major cartilage proteoglycan synthesized by cultured chondrocytes.
J Biol Chem
262:
10229-10238,
1987
4.
Cortes, P,
Riser BL,
Asano K,
Rodriguez-Barbero A,
Narins RG,
and
Yee J.
Effects of oral antihyperglycemic agents on extracellular matrix synthesis by mesangial cells.
Kidney Int
54:
1985-1998,
1998[ISI][Medline].
5.
Day, C.
Thiazolidinediones: a new class of antidiabetic drugs.
Diabet Med
16:
179-192,
1999[ISI][Medline].
6.
Escher, P,
and
Wahli W.
Peroxisome proliferator-activated receptors: insight into multiple cellular functions.
Mutat Res
448:
121-138,
2000[ISI][Medline].
7.
Furnsinn, C,
Neschen S,
Noe C,
Bisschop M,
Roden M,
Vogl C,
Schneider B,
and
Waldhausl W.
Acute non-insulin-like stimulation of rat muscle glucose metabolism by troglitazone in vitro.
Br J Pharmacol
122:
1367-1374,
1997[Abstract].
8.
Helig, CW,
Concepcion LA,
Riser BL,
Freytag SO,
Zhu M,
and
Cortes P.
Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype.
J Clin Invest
96:
1802-1814,
1995[ISI][Medline].
9.
Higashiguchi, T,
Hasselgren PO,
Wagner K,
and
Fischer JE.
Effect of glutamine on protein synthesis in isolated intestinal epithelial cells.
J Parenter Enteral Nutr
17:
307-314,
1993[Abstract].
10.
Isshiki, K,
Haneda M,
Koya D,
Maeda S,
Sugimoto T,
and
Kikkawa R.
Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats.
Diabetes
49:
1022-1032,
2000[Abstract].
11.
Kreisberg, JI,
and
Karnovsky MJ.
Glomerular cells in culture.
Kidney Int
23:
439-447,
1983[ISI][Medline].
12.
Ma, LJ,
Marcantoni C,
Linton MF,
Fazio S,
and
Fogo AB.
Peroxisome proliferator-activated receptor- troglitazone protects against nondiabetic glomerulosclerosis in rats.
Kidney Int
59:
1899-1910,
2001[ISI][Medline].
13.
Masola, B,
Peters TJ,
and
Evered DF.
Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase.
Biochim Biophys Acta
843:
137-143,
1985[ISI][Medline].
14.
McCarthy, KJ,
Routh RE,
Shaw W,
Walsh K,
Welbourne TC,
and
Johnson JH.
Troglitazone halts diabetic glomerulosclerosis by blockade of mesangial expansion.
Kidney Int
58:
2341-2350,
2000[ISI][Medline].
15.
Mongial, M,
Otto J,
Oldershaw R,
Ferrier F,
Sato JD,
and
Iozzo RV.
Fibroblast growth factor-binding protein is a novel partner for perlecan protein core.
J Biol Chem
276:
10263-10271,
2001
16.
Moore, GE,
Gerner RE,
and
Franklin HA.
Culture of normal human leukocytes.
JAMA
199:
519-524,
1967[Medline].
17.
Moreadith, RW,
and
Lehninger AL.
The pathways of glutamate and glutamine oxidation by tumor cell mitochondria.
J Biol Chem
259:
6215-6221,
1984
18.
Nissim, I,
Sahai A,
Sandler R,
and
Tannen RL.
The intensity of acidosis differentially alters the pathways of ammoniagenesis in LLC-PK1 cells.
Kidney Int
45:
1014-1019,
1994[ISI][Medline].
19.
Oda, T,
Funai T,
and
Ichiyama A.
Induction by peroxisome proliferators and triiodothyronine of serine:pyruvate/alanine:glyoxylate aminotransferase of rat liver.
FEBS Lett
418:
265-268,
1997[ISI][Medline].
20.
Peterkofsky, A.
Involvement of glutamic acid and glutamine in protein synthesis and maturation.
In: Enzymes of Glutamine Metabolism, edited by Prusiner S,
and Stadtman E. New York: Academic, 1973, p. 331-342.
21.
Pitts, RF.
Production of CO2 by the intact functioning kidney of the dog.
Med Clin North Am
59:
507-518,
1975[ISI][Medline].
22.
Reitzer, LJ,
Wice BM,
and
Kennel D.
Evidence that glutamine, and not sugar, is the major energy source for cultured HeLa cells.
J Biol Chem
254:
2669-2676,
1979[ISI][Medline].
23.
Riser, RL,
Denichilo M,
Cortes P,
Baker C,
Grondin JM,
Yee J,
and
Narins RG.
Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis.
J Am Soc Nephrol
11:
25-38,
2000
24.
Routh ER, Johnson JH, and McCarthy KJ. Direct action of
troglitazone on mesangial cell behavior: troglitazone suppresses the
secretion of type 1 collagen by mesangial cells in vitro. Kidney
Int. In press.
25.
Schmidt, E.
Glutamate dehydrogenase: UV assay.
In: Methods of Enzymatic Analysis, edited by Bergmeyer H. New York: Academic, 1974, vol. 2, p. 650-655.
26.
Schleicher, ED,
and
Weigert C.
Role of the hexosamine biosynthetic pathway in diabetic nephropathy.
Kidney Int
58:
S13-S18,
2000[ISI].
27.
Schoolwerth, AC,
and
LaNoue KF.
Transport of metabolic substrates in renal mitochondria.
Annu Rev Physiol
47:
143-171,
1985[ISI][Medline].
28.
Spreight, G,
Handley CJ,
and
Lowther DA.
Role of glutamine in glycosaminoglycan synthesis in vitro by chondrocytes.
Biochim Biophys Acta
540:
238-245,
1978[ISI][Medline].
29.
Traxinger, RR,
and
Marshall S.
Coordinated regulation of glutamine: fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine.
J Biol Chem
266:
10148-10154,
1991
30.
Welbourne, TC,
and
Mu X.
Extracellular glutamate flux regulates intracellular glutaminase activity in LLC-PK1-F+ cells.
Am J Physiol Cell Physiol
268:
C1418-C1424,
1995
31.
Welbourne, TC,
Routh RE,
Yudkoff M,
and
Nissim I.
The glutamine/glutamate couplet and cellular function.
News Physiol Sci
16:
157-160,
2001
32.
Wroblewski, F,
and
LaDue JS.
Serum glutamic-pyruvic transaminase in cardiac and hepatic disease.
Proc Soc Exp Biol Med
91:
569-571,
1956.
33.
Xue, HH,
Sakaguchi T,
Fujie M,
Ogawa H,
and
Ichiyama A.
Flux of the L-serine metabolism in rabbit, human and dog livers. Substantial contribution of both the mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.
J Biol Chem
274:
16028-16033,
1999
34.
Zielke, HR,
Ozand PT,
Tildon JT,
Sevdalian DA,
and
Cornblath M.
Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts.
J Cell Physiol
95:
41-48,
1978[ISI][Medline].