From the Diabetes Research Center, Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California.
Address correspondence and reprint requests to Jang H. Youn, PhD, Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, 1333 San Pablo Ave., MMR 626, Los Angeles, CA 90089-9142. E-mail: youn{at}usc.edu .
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ABSTRACT |
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INTRODUCTION |
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One potential mechanism by which impaired glucose metabolism leads to
impairment of insulin's action on Rd involves the
hexosamine biosynthesis pathway (HBP)
(12,15).
The HBP is a minor glucose metabolic pathway converting fructose-6-phosphate
(F-6-P) to nucleotide hexosamines that serve as essential substrates for
protein glycosylation. Marshall et al.
(16,17)
discovered that the HBP is involved in the downregulation of insulin's action
on glucose transport in cultured fat cells exposed to high glucose and insulin
concentrations. A series of experiments by these investigators demonstrated
that increased substrate flux through the HBP results in decreased insulin
action on glucose transport. Subsequently, several groups
(18,19,20)
have demonstrated that an infusion of glucosamine, which increases the HBP
flux, decreases insulin-mediated Rd in vivo by decreasing
insulin's action on GLUT4 translocation in skeletal muscle
(20). The HBP has been
implicated mainly in hyperglycemia-induced insulin resistance ("glucose
toxicity" [21]).
Excessive glucose flux into cells with hyperglycemia would increase
glucose-6-phosphate (G-6-P) and F-6-P levels and consequently increase the HBP
flux by mass action (22).
However, similar changes (i.e., increased G-6-P/F-6-P levels and HBP flux) may
occur with normal glucose influx at euglycemia if intracellular glucose
metabolism is impaired
(12,15).
Thus, the HBP may be involved not only in hyperglycemia-induced insulin
resistance but also in the development of insulin resistance at euglycemia
with metabolic impairment
(12,13).
To support this concept, Hawkins et al.
(15) reported that fat
(Intralipid)-induced insulin resistance was accompanied by two- to threefold
increases in muscle HBP product levels. These data support the notion that
suppression of glycolysis during fat infusion may increase muscle G-6-P/F-6-P
levels and HBP flux to result in insulin resistance. However, these
observations were made with maximally effective insulin concentrations
(3,000 pmol/l), and it is unknown whether similar changes also occur at
physiological insulin concentrations.
The present study was designed to further address the role of the HBP in fat-induced insulin resistance. We examined whether fat infusion (or increased plasma free fatty acid [FFA] levels) at physiological insulin levels increases HBP flux in skeletal muscle, as indicated by HBP product levels. In addition, we examined whether fat-induced insulin resistance is additive to that induced by increased HBP flux via glucosamine infusion and, if so, whether such additive effects correlate with muscle HBP product levels.
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RESEARCH DESIGN AND METHODS |
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Catheterization. At least 4 days before the experiment, the animals
were placed in individual cages with wire floors. The distal one-third of each
rat's tail was drawn through a hole placed low on the side of the cage and
secured there with a rubber stopper
(12,13,14).
This arrangement was required to protect tail blood vessel catheters during
experiments. Animals were free to move about and were allowed unrestricted
access to food and water. Two tailvein infusion catheters were placed the day
before the experiment, and one tail-artery blood sampling catheter was placed
at least 3 h before the start of insulin infusion (i.e., 0700 h).
Catheters were placed percutaneously during local anesthesia with lidocaine
while rats were restrained in a towel. The animals were returned to their
cages after catheter placement with their tails secured as described above and
were free to move about during the experiments. Patency of the arterial
catheter was maintained by a slow (0.016 ml/min) infusion of heparinized
saline (10 U/ml).
Experimental protocols. Two separate studies were performed in
normal rats after an overnight fast (food was removed at 1700 h on the day
before the experiment, and experiments were started at 1100 h).
Study 1: Effects of Intralipid and/or glucosamine infusions on
insulin-stimulated glucose fluxes and muscle HBP product levels.
Hyperinsulinemic-euglycemic clamp was conducted for 480 min with a continuous
infusion of porcine insulin (Novo Nordisk, Princeton, NJ) at a rate of 22 pmol
· kg-1 · min-1 to raise plasma insulin
within a physiological range. Blood samples (30 µl) were collected at 10-
to 20-min intervals for the immediate measurement of plasma glucose, and 20%
dextrose was infused at variable rates to maintain plasma glucose at basal
concentrations (5.6 mmol/l). After the initial 150-min clamp (control
period), the clamps were continued with a constant infusion of saline
(n = 8), Intralipid (Liposyn II [Abbott, North Chicago]; triglyceride
emulsion, 20% wt/vol; 0.9 ml/h) and heparin (40 U/h with 10 U as a priming
bolus; n = 9), glucosamine (30 µmol · kg-1
· min-1; n = 8), or Intralipid and glucosamine
together (n = 8) during the remaining 330 min (treatment period). To
estimate insulin-stimulated whole-body glucose fluxes, we infused
[3-3H]glucose (high-performance liquid chromatography
[HPLC]-purified; Du Pont, Boston, MA) at a rate of 0.2 µCi/min throughout
the clamps. Blood samples for the measurement of plasma 3H-glucose
(60 µl) were taken every 10 min during the last 30 min of the control and
the treatment periods. Additional blood samples (20 or 60 µl) were taken at
0, 10, 30, 90, 150, 160, 180, 240, 360, and 480 min for the determination of
plasma FFA and/or insulin concentrations. At the end of clamps, animals were
anesthetized with pentobarbital sodium injection. Within 5 min, three muscles
(soleus, tibialis anterior, and extensor digitorum longus [EDL]) were taken
from each hindlimb for measurements of HBP products. Each muscle, once
exposed, was dissected out within 2 s, frozen immediately using liquid
N2-cooled aluminum blocks, and stored at -80°C for later
analysis. Glucose and insulin infusions were continued to prevent any
perturbation of glucose metabolism during the muscle sampling procedure.
Study 2: Effects of short Intralipid infusions on muscle HBP product levels. This study was carried out to examine the effects of short-term (50 and 180 min vs. 330 min in study 1) Intralipid infusion on muscle HBP product levels. For this, we used muscles collected from an independent study in which experimental conditions were identical to those of study 1, except for the rate and duration of Intralipid infusion; the Intralipid infusion rate was 0.75 ml/h, slightly lower than the rate in study 1 (i.e., 0.9 ml/h), and muscles were collected 50 and 180 min after the start of Intralipid infusion.
Analysis. Plasma glucose was analyzed during the clamps using 10 µl plasma by a glucose oxidase method on a Beckman glucose analyzer II (Beckman, Fullerton, CA). Plasma insulin was measured by radioimmunoassay using a kit from Linco Research (St. Charles, MO). Plasma FFA was determined using an acyl-CoA oxidasebased colorimetric kit (Wako Pure Chemical Industries, Osaka, Japan). For the determination of plasma 3H-glucose, plasma was deproteinized with ZnSO4 and Ba(OH)2, dried to remove 3H2O, resuspended in water, and counted in scintillation fluid (Ready Safe; Beckman) on a Beckman scintillation counter.
Muscle contents of HBP products (uridinediphospho-N-acetylglucosamine [UDP-GlcNAc] and uridinediphospho-N-acetylgalactosamine [UDP-GalNAc]) were measured using two sequential chromatographic separations with ultraviolet detection basically as described by Rossetti and colleagues (15,23). Frozen muscles were homogenized in three volumes of ice-cold 0.3 mol/l perchloric acid and centrifuged for 5 min at 3,000g at 4°C. The supernatant was then mixed with two volumes of freon (1:4 trioctyl-amine:1,1,2 trichlorotrifluoroethane) and centrifuged for 5 min at 3,000g at 4°C (24). The aqueous phase, cleared of perchloric acid, was mixed with a small volume of 150 mmol/l KH2PO4 (pH 2.5) to have a final concentration of 10 mmol/l. A small amount of tritiated UDP-GlcNAc was added to determine the recovery of muscle UDP-GlcNAc in the subsequent chromatographic procedures. The tissue extract was then run through a 1-ml-strong anion-exchange column (Supelco LC-SAX; Supelco, Bellefonte, PA) for partial purification of HBP products (23). The column was washed with 2 ml of 10 mmol/l KH2PO4 followed by 1 ml of 50 mmol/l KH2PO4. HBP products were eluted with 150 mmol/l KH2PO4 into five separate fractions of 0.2- to 1.0-ml volumes. UDP-GlcNAc and UDP-GalNAc coelute with UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) from the column. Two of these fractions with highest concentrations were combined and injected into HPLC for separation of UDP-sugars. HPLC analysis was carried out on a Beckman HPLC system (Beckman, Fullerton, CA) using a reversephase, ion-pairing isocratic method with two LC18T reverse-phase columns (Supelco) connected in series (23). The columns were equilibrated in mobile phase buffer (125 mmol/l KH2PO4, 5 mmol/l tetrabutylammonium sulfate [TBS], adjusted to pH 6.5 with 125 mmol/l K2HPO4, 5 mmol/l TBS buffer) for 1 h at 1 ml/min before each injection. Samples were run isocratically at 1 ml/min for 35 min with 100% mobile phase buffer followed by 15 min of 60% methanol gradient.
Calculations. Rates of total glucose appearance and whole-body Rd were determined as the ratio of the [3-3H]glucose infusion rate (disintegrations per minute [dpms] per minute) to the specific activity of plasma glucose (dpms per micromole) during the final 30 min of the control and treatment periods. Hepatic glucose output (HGO) was determined by subtracting the glucose infusion rate (GINF) from the total glucose appearance.
Statistical analysis. Data are expressed as means ± SE. The significance of the differences in mean values among different treatment groups was evaluated using the one-way analysis of variance, followed by ad hoc analysis using Tukey's test. The significance of the effects of treatment within the groups was evaluated using the paired t test. P < 0.05 was considered statistically significant.
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RESULTS |
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Fasting plasma glucose (5.9 mmol/l) and FFA (
0.70 mmol/l)
concentrations were similar among the four experimental groups. Plasma insulin
was raised to and maintained at
550 pmol/l during the control period
(0-150 min; Fig. 1A).
During the treatment period (150-480 min), plasma insulin was not
significantly altered with the saline infusion but was increased 20-30% with
the individual and the combined infusions of Intralipid and glucosamine
(P < 0.05). Plasma glucose was clamped at
5.6 mmol/l, similar
to basal levels, in all groups throughout the experiments
(Fig. 1B). Plasma FFA
concentrations decreased similarly in all groups during the control period
(Fig. 1C). During the
treatment period, plasma FFA remained suppressed with the saline and the
glucosamine infusions but were raised to levels (
1.5 mmol/l) above the
basal levels with the infusion of Intralipid (alone or combined with
glucosamine infusion). GINFs required to maintain plasma glucose increased
rapidly during the initial 90 min and reached steady-state levels during the
control period (Fig.
1D). During the treatment period, GINFs were constant
with saline infusion (i.e., control group) but decreased with the Intralipid
and/or glucosamine infusions, with more rapid and profound effects observed
with the Intralipid infusions.
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GINFs decreased 52 and 34% at the end (final 30 min) of the Intralipid and the glucosamine infusions, respectively (P < 0.05 vs. control period; Fig. 2A). When Intralipid and glucosamine infusions were combined, GINFs decreased further (i.e., 72%; P < 0.05 vs. the decreases with the Intralipid or glucosamine alone). Thus, the effects of Intralipid and glucosamine infusions to reduce GINFs were additive. Similarly, insulin-stimulated whole-body Rd decreased 38 ± 2 and 28 ± 3% with the Intralipid and the glucosamine infusions, respectively (P < 0.05 vs. control period; Fig. 2B), and further decreased when the infusions were combined (i.e., 47 ± 1%; P < 0.05 vs. the decreases with the Intralipid or glucosamine alone). HGO was completely suppressed in all groups during the control period and with the saline and the glucosamine infusions during the treatment period. In contrast, HGO was not completely suppressed by insulin in the Intralipid-infused groups (i.e., with elevated plasma FFA levels). Hepatic insulin resistance, reduced ability of insulin to suppress HGO, was more severe when Intralipid was infused together with glucosamine (P < 0.05; 38 ± 3 vs. 22 ± 3 µmol · kg-1 · min-1 with Intralipid alone; Fig. 2C). Thus, glucosamine potentiated FFA induction of hepatic insulin resistance.
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The levels of HBP products (i.e., UDP-GlcNAc and UDP-GalNAc) in skeletal muscles (soleus, EDL, and tibialis anterior) were increased by four- to fivefold with the glucosamine infusion, as expected (Table 1). UDP-hexoses (i.e., UDP-Glc and UDP-Gal) showed a tendency to decrease with the glucosamine infusion. In contrast to the dramatic increases with the glucosamine infusion, muscle HBP product levels were not altered by the Intralipid infusion. Thus, the 30-40% decreases in insulin-stimulated Rd with the Intralipid infusion were accompanied by absolutely no change in muscle HBP product levels (Fig. 3). Also, when infused with glucosamine, Intralipid decreased insulin-mediated Rd below that with glucosamine alone without changing HBP product levels. These data indicate that the HBP was not responsible for fat-induced insulin resistance under our experimental conditions. UDP-glucose levels were increased with the Intralipid infusion in soleus but not in the other muscles. This increase may represent a type I error, since the increase was largely due to two soleus samples in the Intralipid infusion group, of which UDP-glucose levels were significantly higher than the rest of the group.
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Study 2: Effects of short intralipid infusions on muscle HBP product
levels. In the study above, the effects of Intralipid infusion on muscle
HBP product levels were studied after a prolonged (330 min) infusion. It is
possible that HBP product levels increased during an earlier period of the
Intralipid infusion but returned to control levels when insulin resistance was
fully developed after the prolonged infusion. To test this possibility, we
examined muscle HBP product levels on muscles collected after a 50- or 180-min
Intralipid infusion. For this, we used muscles collected from an independent
study in which experimental conditions were identical to those in study 1,
except for the rate and the duration of Intralipid infusion; the infusion rate
was 0.75 ml/h in this study, instead of 0.9 ml/h as in study 1. Intralipid
infusion at this lower rate decreased insulin-stimulated
Rd by 25% within 180 min (data not shown). However,
this effect of Intralipid on insulin-stimulated Rd was
accompanied by no significant changes in HBP product levels in muscles
collected at 50 and 180 min after the start of Intralipid infusion
(Fig. 4). These data further
support the notion that Intralipid infusion induces peripheral insulin
resistance without increasing muscle HBP product levels.
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DISCUSSION |
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Our results are contrary to the findings of Hawkins et al.
(15) that indicated that fat
infusion significantly (two- to threefold) increased muscle HBP product
levels. This increase was shown to be similar in magnitude to those seen
during the development of insulin resistance of similar magnitude with agents
that increase HBP flux (i.e., glucosamine, glucose, and uridine). The reason
for this apparent discrepancy between the two studies is unclear. Fat infusion
rates and plasma FFA levels during fat infusion were similar and therefore
cannot explain the discrepancy. Also, although different muscles were examined
in these studies, the discrepancy cannot be attributed to the differences in
muscle fiber type, since our finding was consistently observed in several
muscles (soleus, EDL, and tibialis anterior) with different fiber
compositions. However, there are other major differences in experimental
conditions between the studies, and the discrepancy might arise from these
differences. First of all, the study of Hawkins et al. was carried out at
maximally effective insulin concentrations (3,000 pmol/l), whereas the
present study was carried out at physiological (
550 pmol/l) insulin
concentrations. The effect of fat infusion (i.e., metabolic suppression) to
increase muscle G-6-P (and its mass action to increase F-6-P and HBP flux)
could have been greater in the Hawkins et al. study because of greater
Rd into muscle at maximally effective insulin
concentrations. To support this idea, our previous study
(25) showed that muscle G-6-P
levels increased during fat infusion to levels that were significantly
(40-50%) higher at maximal than at physiological insulin levels. Thus, it may
be possible that fat infusion significantly increases HBP flux at maximal but
not at physiological insulin concentrations. However, our preliminary data do
not support this possibility (data not shown), and this issue remains
unresolved and merits further investigation. Another difference between the
two studies was the fasting state; 6-h-fasted rats were used in the study of
Hawkins et al., whereas overnight-fasted rats were used in the present study.
Nelson et al. (24) showed that
the activity of glutamine F-6-P amidotransferase (GFAT), the rate-limiting
enzyme of the HBP, in rat skeletal muscle decreased by 30% after an 18-h fast.
These data suggest the possibilitythough unlikelythat the lack
of the effect of fat infusion on muscle HBP product levels in the present
study might be due to decreased GFAT activity in muscles of overnight-fasted
animals. Finally, it may be worthwhile to point out that Sprague-Dawley rats
were used in the study by Hawkins et al., whereas Wistar rats were used in the
present study. There is evidence for differential regulation of glucose
metabolism between the two rat strains. For example, different effects of
aging (or growth) were observed on insulin sensitivity of glucose transport,
lactate production, and glycogen synthesis
(26). It would be extremely
interesting if the discrepancy regarding the role of HBP in fat-induced
insulin resistance arose from the strain difference. Whatever the reason for
the discrepancy, the present data indicate that there was a mechanism
independent of HBP product levels that induced insulin resistance in skeletal
muscle with increased availability of plasma FFA.
The role of the HBP in the regulation of insulin action has been extensively studied since the discovery of Marshall et al. (16,17) that the HBP is involved in the downregulation of insulin action on glucose transport in cultured fat cells exposed to high glucose and insulin concentrations. Increasing HBP flux via glucosamine infusion/treatment has been shown to induce insulin resistance, accompanied by impairment of insulin action on GLUT4 translocation in insulin-sensitive cells in vivo (18,19,20) and in vitro (22,27). In addition, overexpression of GFAT in muscles of transgenic mice (28) or in Rat-1 fibroblasts (29) resulted in impaired insulin action on glucose uptake/transport. These studies have established that increased HBP flux leads to impairment of insulin action on glucose transport in insulin-sensitive cells. The HBP appears to be involved in the development of insulin resistance secondary to hyperglycemia (22,30). However, whether the HBP is also involved in the development of insulin resistance at euglycemia under pathophysiological conditions (e.g., increased plasma FFA levels) has not been rigorously studied. Because insulin resistance develops long before the frank onset of type 2 diabetes (31), this issue may be a critical one in evaluating the role of the HBP in the pathogenesis of type 2 diabetes. HBP product levels and GFAT activity have been shown to be altered in various metabolic states characterized by altered insulin action, including fasting (24), calorie restriction (32), diabetes (22,30), obesity (33), and growth hormone deficiency (34). In addition, GFAT activity, measured in cultured human muscle cells, was inversely correlated with insulin-stimulated Rd in vivo in normal subjects (35). Although these data are consistent with the role of the HBP in the regulation of insulin action at euglycemia, the causal relationship in the association between GFAT activity/HBP products and insulin action remains to be tested.
The HBP has been proposed to serve as a negative feedback control mechanism that senses hyperglycemia or excessive glucose influx to adjust insulin's action on glucose entry (17). To support this idea, it has been demonstrated that hyperglycemia (22) or overexpression of GLUT1 in skeletal muscle of transgenic mice (36) results in insulin resistance associated with increased HBP product levels. Regarding this issue, an important factor in the development of insulin resistance may be the magnitude and/or duration of enhanced Rd into muscle rather than plasma glucose concentration per se. Theoretically, the role of the HBP can be extended to the sensing of the balance between glucose influx and glucose metabolism. G-6-P and F-6-P can be increased not only by increased glucose flux into cells at hyperglycemia but also by suppressed glucose metabolism (e.g., glycolysis) with normal glucose flux into cells at euglycemia, as previously demonstrated (13,15). On the basis of this reasoning, we previously speculated on the potential role of the HBP as part of the mechanism by which metabolic impairment leads to insulin resistance (12). This intriguing possibility was supported by the study of Hawkins et al. (15) but not by the present study. It is unclear whether this discrepancy arose from the difference in the magnitude of glucose flux into muscle, which would have been greater in the Hawkins et al. study because of the higher insulin levels, as discussed in the second paragraph of the DISCUSSION section. Further studies are required to resolve this discrepancy and, more important, to address the issue of whether the HBP plays a role in the acute or chronic development of insulin resistance at euglycemia under various pathophysiological conditions.
We measured HBP product levels as an indicator of HBP flux as in other studies (15,22,24). This would be a reasonable approach if cellular utilization of HBP products is constant or unaltered under the experimental conditions studied. There is little information available on the intracellular kinetics of HBP products, and it is unknown whether cellular utilization of HBP products is subject to regulation by increased FFA levels. Because we cannot exclude the possibility that the Intralipid infusion increased cellular utilization of HBP products (and masked its possible effect to increase HBP flux), our finding of the lack of Intralipid effect on HBP product levels may not be taken as directly indicating a lack of Intralipid effect on HBP flux. However, a significant effect of Intralipid on cellular utilization of HBP products is unlikely, because Intralipid also failed to change HBP product levels raised by glucosamine. Thus, it may be reasonable to conclude that the Intralipid infusion did not increase substrate flux through the HBP under our experimental conditions.
Robinson et al. (22)
suggested that substrate flux through the HBP should be critically dependent
on the level of F-6-P, based on the finding that the apparent
Km for F-6-P of GFAT from muscle (2.4 mmol/l) was
considerably greater than cellular F-6-P levels. Our previous study
(13) showed that muscle G-6-P
levels increased 40% in soleus and EDL muscles during an early phase of
Intralipid infusion under the conditions similar to those of the present
study. Assuming that there were similar increases in F-6-P level, these data,
taken together with the present data, suggest that the HBPan important
metabolic pathway that would affect glycosylation (and thus function) of
various cellular proteinsmay not be significantly altered by
fluctuations in substrate levels and may be regulated rather tightly by other
factors. This concept is supported by the finding of Castle et al.
(37) that three- to fivefold
increases in G-6-P level, induced by increased glycogenolysis with amylin,
failed to increase HBP product levels in perfused rat hindlimb muscles.
In summary, increased availability of plasma FFA at physiological insulin levels induced marked peripheral insulin resistance in overnight-fasted rats, which was accompanied by no change in skeletal muscle HBP product levels. In addition, the FFA-induced insulin resistance was additive to that induced with increased HBP flux via glucosamine infusion, but the additive effects of FFA and glucosamine could not be explained by muscle HBP product levels. Taken together, these data indicate that the HBP was not involved in fat-induced insulin resistance in overnight-fasted rats and that there must be another mechanism by which suppression of glucose metabolism (i.e., glycolysis with fat infusion) leads to impaired insulin action on Rd.
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ACKNOWLEDGMENTS |
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We are grateful to Dr. Luciano Rossetti for helping us to set up the HPLC protocol for the measurement of muscle HBP products. Also, we thank Drs. Chin K. Sung and Joyce M. Richey for their insightful comments on the manuscript.
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FOOTNOTES |
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Received for publication June 12, 2000 and accepted in revised form October 23, 2000
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REFERENCES |
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