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Department of Medicine, University of Mississippi Medical Center, and Veterans Administration Medical Center (E.D.C.), Jackson, Mississippi 39216
Address all correspondence and requests for reprints to: Errol D. Crook, M.D., Division of Nephrology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216. E-mail: ecrook{at}medicine.umsmed.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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8-fold) in cells cultured in increasing glucose
concentrations (1–20 mM). There was a shift to the left in
the glucose dose-response curve for glycogen content in GFA cells
(ED50 for glycogen content = 5.80 ± 1.05
vs. 8.84 ± 0.87 mM glucose, GFA
vs. control). Inhibition of GFA reduced glycogen content
by 28.4% in controls cultured in 20 mM glucose. In a
dose-dependent manner, glucose resulted in a more than 35% decrease in
GP activity in controls. GP activity in GFA cells was suppressed
compared with that in controls, and there was no glucose-induced
down-regulation of GP activity. Glucosamine and uridine mimicked the
effects of glucose on glycogen content and GP activity. However,
chronic overexpression of GFA is a unique model of hexosamine excess,
as culturing control cells in low dose glucosamine (0.1–0.25
mM) did not suppress GP activity and did not eliminate the
glucose-mediated down-regulation of GP activity. We conclude that
increased flux through the HBP results in enhanced glycogen
accumulation due to suppression of GP activity. These results
demonstrate that the HBP is an important regulator of cellular glucose
metabolism and supports its role as a cellular glucose/satiety sensor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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How excess glucose leads to these effects is not completely understood, but several theories have been investigated. One potential mechanism by which excess glucose may exert its effects is via metabolism through the hexosamine biosynthesis pathway (HBP) (7, 8). This pathway, that accounts for 2–3% of cellular glucose flux converts fructose-6-phosphate to glucosamine-6-phosphate by the transfer of an amide group from glutamine. The first and rate-limiting step in this pathway is catalyzed by the enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). The HBP has been shown to mediate glucose disposal (9, 10, 11), insulin resistance (12, 13, 14, 15), and growth factor regulation (16). For example, rats infused with glucosamine demonstrated decreased glucose disposal (9) that is mediated by decreased GLUT4 (glucose transporter) translocation (10). In addition, transgenic mice overexpressing GFA in skeletal muscle and fat have decreased insulin stimulated glucose disposal (11) that is mediated in part by decreased GLUT4 translocation (17).
We have shown that overexpression of GFA in Rat-1 fibroblast renders them insulin resistant (12, 13). This insulin resistance was manifested by a shift to the right in the insulin dose-response curve for glycogen synthase (GS) activity. In addition, overexpression of GFA made the cells more sensitive to the effects of glucose (13). In Rat-1 cells, basal and insulin-stimulated GS activities were decreased in high glucose and GFA overexpression enhanced these effects of glucose. In this work we aimed to test the effects of excess hexosamines on glycogen metabolism. Given our previous data, we hypothesized that overexpression of GFA would result in decreased glycogen content at low glucose concentrations. Using Rat-1 fibroblasts that overexpress GFA, we show that metabolism of glucose through the HBP is responsible at least in part for the regulation of glycogen content, and this regulation of glycogen content is mediated via hexosamine-mediated effects on glycogen phosphorylase (GP) activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-14C]Glucose-1-phosphate was
purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA).
Routine reagents were purchased from Sigma (St. Louis, MO)
unless otherwise indicated.
Cell culture
Rat-1 fibroblasts that do and do not (control) stably
overexpress the human complementary DNA for GFA were used for these
studies. Cells were cultured in DMEM supplemented with 10% FCS and 0.5
mg/ml gentamicin and routinely passaged at confluence every 4 days
using 10-cm culture dishes. Eighty-five to 90% confluent monolayers
were incubated in DMEM supplemented with 0.2% FCS and the desired
concentrations of glucose, glucosamine, or uridine. The protein
concentration in cell extracts was determined by the method of Bradford
(18) using BSA as the standard.
Glycogen content
Cells were cultured in the indicated glucose and/or
glucosamine/uridine concentrations. Plates were washed twice with
ice-cold PBS and harvested by scraping and pipetting into a 1.5-ml
microfuge tube. Cells were pelleted by centrifuging at 14,000 rpm for 1
min. The supernatant was aspirated, and the weight of the cell pellet
was determined and then frozen in liquid nitrogen. Glycogen content was
determined with amyloglucosidase according to the method of Keppler and
Decker (19). Cell pellets were sonicated in 5 parts by weight of
ice-cold 0.6 N perchloric acid. After centrifugation for 15
min, 0.2 ml of the homogenate was incubated with 0.1 ml 1 M
potassium hydrogen carbonate and 2.0 ml 10 mg/ml amyloglucosidase in a
stoppered centrifuge tube with shaking at 40 C for 2 h. The
reaction was stopped by the addition of 1.0 ml 0.6 N
perchloric acid. The samples were centrifuged for 15 min, and 100 µl
acid supernatant were used for determination of glucose in the presence
of triethanolamine buffer [0.3 M triethanolamine (pH 7.5),
0.5 mM MgSO4, 1 mM ATP, 0.9 mM
NADP, and 5 µg glucose-6-phosphate dehydrogenase] and 5 µl 2 mg/ml
hexokinase. The glucose liberated after hydrolysis of glycogen is
proportional to the increase in NADPH measured by the extinction change
at 340 nm. Glycogen content is expressed as milligrams of glycogen per
g wet wt of cells.
Glycogen phosphorylase activity assay
Glycogen phosphorylase activity was determined by the
incorporation of [U-14C]glucose-1-phosphate
into glycogen in the presence and absence of the allosteric activator
AMP (5 mM) (20). Cells were harvested by scraping into a
buffer containing 10 mM Tris-HCl (pH 7.0), 150
mM potassium fluoride, 15 mM EDTA, 15
mM 2-mercaptoethanol, 10 µg/ml leupeptin, 1
mM benzamidine, and 1 mM
phenylmethylsulfonylfluoride. The reaction was initiated by the
addition of 10 µl extract to 20 µl reaction mixture containing 200
mM KF, 95 mM glucose-1-phosphate, 5
mM [U-14C]glucose-1-phosphate (50
µCi/mmol), and 3.4 mg/ml glycogen with or without 5 mM
AMP. After 20 min at 30 C, 25 µl were spotted onto
Whatman filter paper (Clifton, NJ) and washed in 66%
ethanol for 20 min for a total of four washes. The papers were
air-dried, and radioactivity was determined. Fractional activity of GP
was expressed as the ratio of activity in the absence of AMP to that in
the presence of AMP (total activity).
UDP-N-acetyl-glucosamine
Levels of uridine diphospho-N-acetyl-glucosamine
(UDP-GlcNAC) were determined in cells using a modification of the
procedure described by Robinson et al. (21). Cells were
harvested into the same buffer used for phosphorylase assays (see
above) and centrifuged at 14,000 rpm for 1 min. The supernatant was
aspirated, and the pellet was resuspended in 200 µl of the same
buffer and frozen in liquid nitrogen. Just before the assay the cells
were thawed, and the pellet was crudely resuspended and sonicated.
dH2O (467 µl) and 333 µl 1
M perchloric acid (final concentration, 0.333
M in a 1-ml final volume) were added. The tube
was vortexed and centrifuged at 14,000 rpm for 8 min at 4 C. One
milliliter of the supernatant was then placed into a 2-ml centrifuge
tube. Nine hundred microliters of
trioctylamine:1,1,2-trichlorofluoethane (1:4) were added, and the tube
was vortexed and centrifuged at 14,000 rpm for 4 min at 4 C.
Supernatant (100 µl) was added to 650 µl 5 mM
KH2PO4 and mixed, and the
entire volume (750 µl) was loaded onto the HPLC. HPLC was performed
on a Spherisorb amino column (25 cm x 4.6 mm; Phase Separations,
Inc., Hesperia, CA) eluted with a concave gradient from 80
mM
KH2PO4 (pH 2.8) and 35%
acetonitrile to 800 mM
KH2PO4 (pH 3.6) over 48 min
at a flow of 1 ml/min. UDP-GlcNAC levels were quantified by UV
absorption at 254 nm compared with external standards and corrected for
protein amount of the original extract.
Glucosamine-6-phosphate (GlcN-6-P)
GlcN-6-P levels were determined using a modification of
the previously described procedure for determining GFA activity (22).
Cells were harvested as described for phosphorylase assays and frozen
in liquid nitrogen. On the day of the assay the pellets were thawed on
ice, sonicated, and centrifuged at 14,000 rpm for 2 min at 4 C. One
hundred microliters of the supernatant were placed into a 1.5-ml
microfuge tube and 0.5 vol 1 M perchloric acid was added.
The tube was vortexed, placed on ice for 10 min, and centrifuged at
14,000 rpm for 10 min at 4 C. Supernatant (145 µl) was taken, and 258
µl trioctylamine-1,1,2-trichlorofluoethane (1:4) were added. The
mixture was vortexed and centrifuged at 14,000 rpm for 1 min. One
hundred and twenty microliters of the aqueous phase were derivatized
with 2 vol o-phthaldialdehyde (OPA) solution (4 mg OPA
dissolved in 50 µl ethanol and added to 5 ml 0.1
M sodium borate and 10 µl 2-mercaptoethanol)
for 1 min at room temperature. The samples were then neutralized with
330 µl 0.1 M sodium phosphate (pH 7.4),
filtered, and separated over a reverse phase C18
column (25 cm x 4.6 mm; Spherisorb ODS, Phase Separations, Inc.).
Absorbance of the sample eluent was analyzed fluorometrically at 340 nm
(excitation) and 460 nm (emission), and peak areas were integrated. OPA
GlcN-6-P standards were run separately to determine retention time and
generate a standard curve to correlate area to the amount of
GlcN-6-P.
Statistical methods
Data are expressed as the mean ± SEM.
Student’s t test was used for determining statistical
significance between means, and P 
0.05 was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Control and GFA cells were glycogen depleted by
culture in 1 mM glucose [low glucose (LG)]
overnight (16–24 h). Cells were then refed with 1–20
mM glucose for 8–9 h, and glycogen content was
determined. Both control and GFA cells demonstrated a dose-dependent
increase in glycogen content when cultured in increasing concentrations
of glucose (Fig. 1A). The accumulation of
glycogen in GFA cells was more pronounced at lower glucose
concentrations compared with controls. The ED50
for glucose-induced glycogen accumulation in GFA cells was 5.80 ±
0.87 mM, whereas in control cells it was
8.84 ± 1.05 mM (P < 0.05;
Fig. 1B).
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). Culturing
in uridine (0–3 mM), which enters the HBP distal
to glucosamine, lead to a similar dose-dependent, statistically
significant (P < 0.05) increase in glycogen content
(Fig. 2).
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, culturing
cells in DON led to a 28.4% (P = 0.01) decrease in
glycogen content in control cells cultured in 20
mM glucose [high glucose (HG)]. DON had no
effect on glycogen content in cells cultured in LG and/or GlcN. The
decrease in glycogen content in cells cultured in HG with DON (15
mg/g wet wt) is similar to the increase in glycogen content seen in
cells cultured in GlcN.
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).
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). Similarly, GPT activity was increased
2.2-fold (P = 0.02) in GFA cells cultured in 1
mM glucose compared with controls cultured at 1
mM glucose. Compared with 1
mM, 20 mM glucose did not
result in a statistically significant increase in GPT activity in GFA
cells. Specific GP activity in the absence of cAMP (GPa) is also shown
in Table 1. There was a trend toward higher GPa activity in GFA cells
that did not reach statistical significance.
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). There was no difference in total GP
activity noted between cells cultured in HG, glucosamine, or uridine.
Moreover, the regulation of GP by HG and GlcN was also observed in
glycogen-replete cells. Cells were made glycogen replete by culture in
HG overnight, and GP was determined after refeeding with LG, HG, or
LG/GlcN for 9 h. Cells refed with LG demonstrated a 60%
(P < 0.05) increase in GP activity, but there was no
change in activity in cells refed with HG or LG/GlcN (not shown).
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).
In cells cultured in glucosamine, UDP-GlcNAC levels are 10-fold higher
than even the levels seen in HG conditions (P < 0.05).
GFA overexpression results in a 2-fold (P < 0.05)
increase in UDP-GlcNAC levels in cells cultured in LG compared with
controls (Table 2). UDP-GlcNAC levels increased only modestly with HG
in GFA cells, and there was no statistically significant difference in
UDP-GlcNAC levels between controls and GFA cells cultured in HG.
GlcN-6-P levels were not statistically different in cells cultured in
LG, HG, or GlcN (9.55–14.9 nmol GlcN-6-P/mg protein).
Chronic overexpression of GFA has specific effects
The effects of high glucose, glucosamine, and GFA overexpression
on glycogen content and GP activity were seen in conditions of
increased UDP-GlcNAC levels. We performed experiments to determine
whether the effects of GFA overexpression could be duplicated in other
conditions of modest elevations in UDP-GlcNAC. Control cells were
cultured in 0.1–0.25 mM GlcN overnight to achieve modest
elevations of UDP-GlcNAC such as those observed in GFA cells. Cells
were then switched to 0.1 mM GlcN with 1–20 mM
glucose for 8–9 h, and GP activity was determined. Unlike GFA cells,
cells cultured in low dose GlcN still had a dose-dependent
glucose-induced decrease in GP activity (Fig. 6). These results demonstrate that
chronic overexpression of GFA is a unique model of hexosamine excess,
and they support findings that some of the effects of glucosamine may
not be explained by hexosamine flux, but may be due to specific
untoward physiological effects of glucosamine (23).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we investigate the effects of the HBP on the regulation of
cellular glycogen content. We show that glycogen content is increased
by both high glucose and downstream products of the HBP, glucosamine
and uridine. Blockade of GFA, the rate-limiting enzyme in the HBP,
partially inhibits the high glucose-induced increase in glycogen
content. Moreover, overexpression of GFA results in enhanced
glucose-mediated glycogen accumulation, as demonstrated by a shift to
the left in the glucose dose-response curve for glycogen content (Fig. 1). The enhanced glycogen accumulation observed with GFA overexpression
is associated with suppression of GP activity. Similarly, high glucose,
glucosamine, and uridine down-regulate GP activity. In short, GFA cells
behaved as if they were in higher glucose conditions compared with
controls, and products of the HBP mimicked the effects of glucose.
Taken together, these results support the importance of the HBP in
mediating cellular metabolic processes and establish further its role
as a glucose-sensing pathway.
These data extend our previous work examining the effects of excess hexosamines on GS regulation. We have shown that GFA overexpression results in both insulin resistance and increased sensitivity to glucose. For example, in GFA cells, there was a shift to the right in the insulin dose-response curve for insulin-stimulated GS activity (12, 13). Secondly, both basal and insulin-stimulated GS activity were decreased by high glucose in a dose-dependent fashion, and overexpression of GFA made cells more sensitive to these effects of glucose. Interestingly, in this work we observed the highest glycogen content in conditions where GS activity was previously shown to be decreased, with HG and GlcN. As this is counterintuitive, that effect of HG and GlcN on GS activity was confirmed in these studies. HG and GlcN again resulted in a more than 70% decrease in GS activity (not shown). It is not entirely understood why we see a decrease in GS activity in conditions where glycogen is accumulating. This paradox suggests that glycogen per se may have a regulatory role on the activation state of GS. The down-regulation of GS activity seen with high glucose or glucosamine required 6 h of culture and became maximal at 12 h (13). In the current studies glycogen content was determined 8–9 h after culture in high glucose or glucosamine. Therefore, the high glucose-induced down-regulation of GS activity is temporally related to the increase in glycogen content, supporting a role for glycogen regulation of GS activity. Another possibility is that GP, not GS, is the major determinant of glycogen content in Rat-1 cells cultured in high glucose or glucosamine for several hours. However, it is not clear from the results presented here whether glycogen has a regulatory role in GP activity. Ongoing studies are examining these questions.
Some of the effects of hexosamine synthesis on glycogen content may be partially dependent on substrate availability. For example, 3 mM glucosamine results in less glycogen accumulation that 20 mM glucose, whereas in most other studies glucosamine is more potent than glucose in producing its effects. Secondly, inhibition of GFA with DON reduces glycogen accumulation by only 25% in HG conditions, and the absolute amount of this decrease in glycogen content is similar to the increase in glycogen content seen with glucosamine and uridine. Thirdly, culture in glucosamine leads to much larger UDP-GlcNAC levels than even HG, arguably enlarging the UDP pool for synthesis of UDP-glucose, the substrate for glycogen synthesis. Finally, 3 mM uridine, which could feed directly into the UDP pool, resulted in glycogen content similar to that under HG conditions.
Nevertheless, the shift to the left in the glucose dose-response curve for glycogen content seen in GFA cells supports a regulatory role for the HBP beyond that of increased substrate for glycogen synthesis. For instance, if substrate availability were the only factor determining increased glycogen content in GFA cells, we would expect glycogen levels to be different between control and GFA cells at 1 mM glucose. UDP-GlcNAC levels are 2-fold elevated in GFA cells cultured at 1 mM glucose compared with control values, yet there is no difference in glycogen content at that glucose concentration. Secondly, the suppression of GP activity in GFA cells points to a mechanism by which the HBP may regulate glycogen content.
Other investigators have used glucosamine infusion as a model for excess hexosamine flux, but they have not found skeletal muscle GP and GS activities to be affected (9). The difference between the data presented here and the above-mentioned studies may be explained by differences between the chronic and acute effects of hexosamines. As mentioned above, it requires several hours in culture for the effects of glucose and hexosamines on GS regulation to occur. In the present studies the down-regulation of GP activity by high glucose required at least 2–4 h in culture (not shown). The relatively short period of glucosamine infusion used in the studies mentioned above may have been insufficient for the effects on GS and GP to occur. In addition, the difference in tissue (skeletal muscle vs. fibroblasts) may account for the varied results.
It has been shown that some of the effects of glucosamine may be due to nonphysiological effects such as ATP depletion (23). We have demonstrated that the effects of glucosamine are generalizable to other products of the HBP, as uridine increased glycogen content and down-regulated GP activity similar to glucosamine. We also demonstrate a difference in cells cultured in glucosamine compared with another model of hexosamine excess, GFA overexpression. Unlike GFA cells, cells cultured in low dose glucosamine continue to demonstrate glucose-mediated down-regulation of GP activity. Therefore, chronic, stable changes in hexosamine metabolism may result in important mechanistic and perhaps phenotypic differences compared with models using acute and subacute exposure to glucosamine.
How GP activity is regulated by the HBP is not completely understood. The increase in total (cAMP-dependent, GPT) GP activity with HG, GlcN, and GFA overexpression indicates an increased enzyme mass in these conditions. Therefore, the HBP may regulate GP at a transcriptional level. This idea is supported by the finding that the HBP regulates transcription of growth factors (16, 26) and transcription factors (27). We have not observed this effect on total GS activity (glucose-6-phosphate dependent) (12, 13), and it appears that regulation of GS by the HBP is via changes in its phosphorylation (activation) state. To this end we have evidence that hexosamine excess regulates several of the kinases (28) and the phosphatase (14) that determine the phosphorylation state of GS. Like GS, the changes in fractional activity of GP presented here are presumptively due to changes in its phosphorylation state. GP exists in an inactive dephosphorylated (GP b) form and an active phosphorylated (GP a) form. Although GPT increased with glucose, glucosamine, and GFA overexpression, there was no significant change in cAMP-independent GP activity in these conditions. One of the consequences of this decrease in fractional GP activity is glycogen accumulation.
Phosphorylase kinase phosphorylates GP b to convert it to GP a, whereas the type 1 phosphatase does the opposite. In previous work we did not observe an effect of glucose or glucosamine on basal phosphorylase kinase activity. However, hexosamine excess did result in insulin resistance for phosphorylase kinase activity. The inactivation of phosphorylase kinase by insulin was inhibited with both high glucose and glucosamine (28). The lack of effect of glucose and glucosamine on basal phosphorylase kinase activity are not consistent with the current results for GP, as we observed glucose- and hexosamine-induced decreases in basal GP activity. Contrarily, we did not see an effect of insulin on GP activity (data not shown) in these cells. This is not to say that the GP is not under hormonal control. Stimulation of cAMP-dependent protein kinase (protein kinase A) with forskolin (100 µM for 20 min) results in a more than 50% increase in GP activity (n = 3; P < 0.05).
Phosphorylase kinase also phosphorylates (and hence inactivates) GS (29). The effects of hexosamines on phosphorylase kinase correlate with the above-mentioned effects on GS. Therefore, in Rat-1 cells, it appears that the effects of hexosamine excess on phosphorylase kinase may regulate its activity toward phosphorylation of GS rather than GP. Others have demonstrated this discrimination in activity of phosphorylase kinase between GS and GP (30). We are not prepared to conclude, however, that regulation of phosphorylase kinase by the HBP is not involved in regulation of GP, as more specific studies have to be performed. For example, the type 1 glycogen-associated phosphatase is targeted to the glycogen particle by a protein known as the protein targeting to glycogen (31). The protein targeting to glycogen also forms complexes with GS and GP as well as phosphorylase kinase. Ongoing studies, performed in high velocity glycogen particles, are focused on examining the regulation of these enzymes involved in glycogen metabolism. This technique has provided interesting preliminary data on hexosamine regulation of GS targeting, and it is hoped that it will provide more mechanistic information on the regulation of GP and phosphorylase kinase. In addition, it will help to determine whether glycogen regulates GS activity as hypothesized above.
It is important to note that the assays for GP and GS are in vitro assays and may not directly reflect in vivo conditions. The presence of activators or inhibitors, such as glucose-6-phosphate, cAMP, or even UDP-GlcNAC could be important. We do not think that the suppression of GP activity by GFA or HG is explained solely by substrate flux or the presence of soluble inhibitors. This assay is performed under conditions where cytoplasmic extracts are diluted 60- to 100-fold, and therefore, the presence of regulating substances would be greatly diminished. Secondly, desalting of like extracts with Sephadex G-15 columns did not alter the effects of HG or GlcN on GS (12, 13), and the exogenous addition of UDP-GlcNAC had no effect on GS activity in vitro.
We have hypothesized that glucose flux through the HBP is important in cellular sensing of glucose and in regulating many glucose-inducible metabolic effects (8). Recent work has extended the role of the HBP in regulating cellular metabolism. For example, the HBP has now been implicated as a sensor and mediator of fat metabolism (32, 33). In addition, the role of this pathway in mediating diabetes has been supported by evidence that troglitazone may reverse the decrease in glucose disposal induced by overexpression of GFA in skeletal muscle and fat in transgenic mice (17). The results presented here further support that hypothesis. In Rat-1 cells, cellular glycogen content is mediated by GFA and products of the HBP. Unlike skeletal muscle and fat, fibroblasts are neither responsible for significant glucose disposal nor extremely sensitive to insulin. Nevertheless, this system serves as a good model to dissect the mechanisms by which the HBP acts as a cellular sensor of satiety and how it regulates cellular glucose disposal. The results presented here are similar to concurrent work on hepatic glycogen metabolism in transgenic mice overexpressing GFA (34). In those animals that overexpress GFA in liver, an increase in hepatic glycogen content was observed in randomly fed transgenic animals compared with that in controls, and glycogen accumulation was enhanced in transgenics with feeding.
In summary, we have shown that glucose flux through the HBP is important in determining cellular glycogen content. Hexosamine-mediated down-regulation of GP activity is responsible for increased glucose-mediated glycogen content in cells overexpressing GFA. This work supports the importance of the HBP in regulating cellular glucose metabolism and supports its role in the development and course of clinical conditions characterized by altered glucose metabolism such as seen in diabetes mellitus. Supporting this is the observation that GFA activity in skeletal muscle of type 2 diabetics is increased compared with that in controls, and GFA activity increases with hyperglycemia (35, 36).
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Footnotes |
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Received October 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression is mediated by products of the hexosamine
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