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Activation of the Hexosamine Pathway by Glucosamine in Vivo Induces Insulin Resistance in Multiple Insulin Sensitive Tissues¹

Minerva Foundation Institute for Medical Research and Department of Medicine (H.Y.-J.), Division of Endocrinology and Diabetology, University of Helsinki, Helsinki, Finland; and the Veterans Administration Medical Center (M.C.D.) and Department of Medicine (D.M.), University of Mississippi Medical Center, Jackson, Mississippi 39216

Address all correspondence and requests for reprints to: Dr. Antti Virkamäki, Minerva Foundation Institute for Medical Research, Tukholmankatu 2, Helsinki, SF-00250 Finland. E-mail: virkamak{at}helsinki.fi

	Abstract

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We determined the effect of infusion of glucosamine (GlcN), which bypasses the rate limiting reaction in the hexosamine pathway, on insulin-stimulated rates of glucose uptake and glycogen synthesis in vivo in rat tissues varying with respect to their glutamine:fructose-6-phosphate amidotransferase (GFA) activity. Three groups of conscious fasted rats received 6-h infusions of either saline (BAS), insulin (18 mU/kg·min) and saline (INS), or insulin and GlcN (30 µmol/kg·min, GLCN). [3-³H]glucose was infused to trace whole body glucose kinetics and glycogen synthesis, and rates of tissue glucose uptake were determined using a bolus injection of [1-¹⁴C]2-deoxyglucose at 315 min. GlcN decreased insulin-stimulated glucose uptake (315–360 min) by 49% (P < 0.001) at the level of the whole body, and by 31–53% (P < 0.05 or less) in the heart, epididymal fat, submandibular gland and in soleus, abdominis and gastrocnemius muscles. GlcN completely abolished glycogen synthesis in the liver. GlcN decreased insulin-stimulated glucose uptake similarly in the submandibular gland (1.3 ± 0.2 vs. 2.0 ± 0.3 nmol/mg protein·min, GLCN vs. INS, P < 0.05) and gastrocnemius muscle (1.4 ± 0.3 vs. 3.1 ± 0.5 nmol/mg protein·min), although the activity of the hexosamine pathway, as judged from basal GFA activity, was 10-fold higher in the submandibular gland (286 ± 35 pmol/mg protein·min) than in gastrocnemius muscle (27 ± 3 pmol/mg protein·min, P < 0.001). These data raise the possibility that overactivity of the hexosamine pathway may contribute to glucose toxicity not only in skeletal muscle but also in other insulin sensitive tissues. They also imply that the magnitude of insulin resistance induced between tissues is determined by factors other than GFA.

	Introduction

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STUDIES PERFORMED in patients with insulin-dependent diabetes mellitus (IDDM) (1) and in rats in vivo (2) have established chronic hyperglycemia as an independent cause of insulin resistance. Overactivity of the hexosamine pathway has recently been suggested to be one of the possible mechanisms mediating the glucose induced insulin resistance or glucose toxicity (3). GFA is the rate-limiting enzyme of this pathway, which catalyzes the formation of glucosamine-6-phosphate (GlcN6P) and glutamic acid from glutamine and fructose-6-phosphate (4). Overexpression of GFA in transgenic mice (5) and in Rat-1 fibroblasts (6) results in increases in hexosamine concentrations and severe insulin resistance. Hyperglycemia per se is also able to increase both GFA activity and hexosamine concentrations (7). Infusion of glucosamine, which increases intracellular hexosamine concentrations and thereby flux via the hexosamine pathway, by bypassing the reaction catalyzed by GFA, induces insulin resistance in skeletal muscle of normal rats (8, 9). Preexposure to glucosamine also induces insulin resistance in isolated rat diaphragms (10). In diabetic rats, infusion of glucosamine has no effect on insulin-stimulated glucose uptake, suggesting that insulin resistance in these rats is secondary to activation of the hexosamine pathway (9).

GFA activity is found in several tissues other than skeletal muscle in the rat (11, 12, 13). These include insulin-sensitive tissues such as the heart, adipose tissue, the liver, and the submandibular gland (13). The possibility that overactivity of the hexosamine pathway also contributes to insulin resistance in these insulin sensitive tissues has not yet been tested.

Studies in adipocytes have suggested that GFA is under rapid transcriptional control and that its activity can be significantly inhibited by exposing adipocytes to glucosamine or glucose, insulin and glutamine (14). Half-maximal inhibition of GFA activity was observed at a glucosamine concentration of 0.21 mmol/liter within 4 h. However, recent studies using an assay specifically measuring GlcN6P by HPLC rather than a variety of hexosamines by the colorimetric technique (7) have suggested that GFA activity in skeletal muscle is unaffected by 4 h of hyperinsulinemia in normal subjects (15) but can be increased by prolonged exposure to glucose and insulin in cultured human muscle cells (16). Whether these discrepant results regarding regulation of GFA are due to species, tissue or methodological differences, is unknown.

The effect on glucosamine induced insulin resistance on the intracellular fate of glucose is also controversial. In rats in vivo, coinfusion of glucosamine as compared with saline with insulin decreases the rate of glycogen synthesis in rat abdominis muscle. On the other hand, in isolated soleus muscle, glucosamine induces insulin resistance of glucose uptake but preferentially channels glucose to glycogen (17). This effect could be attributed to stimulation of glycogen synthase activity by GlcN6P, which accumulated in glucosamine infused rats (data not shown). However, it is unclear whether the different results regarding the effect of glucosamine on glycogen synthesis can be attributed differences in GlcN concentrations or to the type of muscle examined.

The present study was undertaken to answer several of the above questions. First, we wished to determine whether GlcN induces insulin resistance of glucose uptake in tissues other than skeletal muscle. Second, we determined the effect of in vivo hyperinsulinemia on GFA activity in various insulin-sensitive tissues. Finally, we determined the effect of an in vivo GlcN infusion on the rate of insulin-stimulated glycogen synthesis and the glycogen content in multiple insulin sensitive tissues including three different types of skeletal muscle, the heart, epididymal fat, the liver and the submandibular gland, which is both insulin sensitive (18) and has a several-fold higher GFA activity than skeletal muscle (13).

	Materials and Methods

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Preparation of animals
Male Wistar rats (250–300 g) were purchased from the Helsinki University Experimental Animal Center. The animals were housed in a 12-h light-dark cycle and fed standard rodent chow (Altromin 1323, Feedcon, Helsinki, Finland). Six days before the study, the rats were anesthetized (pentobarbital, 60 mg/kg, ip), and catheters were inserted into the aortic arch for blood sampling and right atrium for infusions as previously described (19).

Study design
Three groups of rats were studied. The first group received only saline but no insulin or GlcN (basal, BAS, n = 6). Two other groups were studied under normoglycemic hyperinsulinemic conditions (insulin infusion rate 18 mU/kg·min). These rats received, in addition to glucose and insulin, iv infusions of either saline (INS rats, n = 8) or GlcN at a rate of 30 µmol/kg·min (GLCN rats, n = 8), as detailed below.

The rats were studied after a 16-h fast. On the study morning (0730 h), the rats were weighed, connected to infusion and blood sampling lines, and placed in metabolic cages, where they were allowed to move freely (19). After 1 h, a primed/continuous infusion of insulin (INS and GLCN rats) or saline (BAS) was started (19, 20, 21). Plasma glucose was measured every 5 min (INS and GLCN rats) or 30 min (BAS) with the glucose oxidase method (22) using Beckman Glucose Analyzer II (Beckman Instruments Corp., Fullerton, CA). In INS and GLCN rats, a variable rate infusion of 20% glucose was started at 4 min and adjusted to maintain the plasma glucose concentration at the fasting concentrations of 5.5 mmol/liter. Serum insulin concentrations at 0, 30, 60, 120, 180, 240, 300, and 360 min were determined using RIA (23).

In all groups, a primed (15 µCi), continuous (0.2 µCi/min) infusion of \[3-³H\]glucose was started at 0 min to measure rates of whole body glucose utilization and glycolysis (20). Plasma samples for determination of glucose ([³H]GSA) and water specific activities (WSA) were withdrawn at 0, 30, 60, 120, 180, 240, 300, and 360 min. At 315 min, a bolus injection of 2-\[1-¹⁴C\]-deoxyglucose (¹⁴C-DOG, 50 µCi) was given to determine tissue glucose uptake. Samples for determination of ¹⁴C-DOG specific activity in plasma (dpm/µmol glucose) were withdrawn at 317, 320, 325, 330, 335, 345, and 360 min. At the end of the study, the rats were anesthetized (pentobarbital, ia) and tissues (soleus, gastrocnemius and abdominis muscles, epididymal fat, liver, heart, and submandibular gland) were freeze-clamped in situ with aluminum tongues precooled in liquid nitrogen for measurement of glycogen and 2-\[1-¹⁴C\]deoxyglucose-6-phosphate ([¹⁴C]DOG6P) concentrations, rates of liver glycogen synthesis, and GFA activities. GFA activity was measured in tissues obtained from the BAS and INS rats but not in GLCN rats as increases in GlcN6P formed from GlcN in vivo (data not shown) interferes with in vitro measurement of GlcN6P formation by GFA. Tissue samples were stored in liquid nitrogen until analysis. The experimental protocol was approved by the Ethical Committee of the Helsinki University Central Hospital.

Plasma glucose and water specific activities
Aliquots of plasma (50 µl) for the determination of [¹⁴C]DOGSA and [³H]GSA and WSA were deproteinized with 100 µl Ba(OH)₂ and 100 µl Zn(SO)₄ and centrifuged. The protein-free supernatant (Somoqyi filtrate) was divided into two aliquots, of which one (40 µl) was counted directly to obtain total [³H]- and [¹⁴C]-radioactivity after adding liquid scintillation fluid (OptiPhase 'HiSafe' 3, Wallac UK, Milton Keynes, UK) in a Rackbeta 1214 liquid scintillation counter (Wallac, Turku, Finland). The other aliquot was counted for [³H]- and [¹⁴C]-radioactivity after evaporation to dryness and reconstitution in water (40 µl). Specific activities were calculated by dividing [³H]- and [¹⁴C]-radioactivities in the dried reconstituted aliquots by the ambient plasma glucose concentration. WSA was calculated by subtracting [³H]-radioactivity of the dried Somoqyi filtrate from total [³H]-radioactivity (20). Plasma water was assumed to be 93% of total plasma volume and total body water mass 65% of the body mass (20).

Glycogen concentration and specific activity
The frozen tissue samples were freeze-dried (Edwards EF4 Modulyo, Edwards High Vacuum, West Sussex, UK), dissected free of blood and connective tissue, weighed, and extracted for 15 min at +50 C with 1 N KOH (100 µl/mg dry) (24). The alkaline extract was used for the determination of tissue glycogen concentration and glycogen specific activity.

Tissue glycogen concentrations were determined after alkaline hydrolysis with amyloglucosidase (25). In brief, an aliquot of the alkaline extract (200 µl) was neutralized with 1 N HCl and its pH was adjusted to 4.9 with 0.15 N sodium acetate buffer. The glucose concentration was determined before and after amyloglucosidase (Sigma Chemicals, St. Louis, MO) treatment (1 h at room temperature) in the acified extract (26). The glycogen concentration (mmol/kg dry) was calculated by dividing the difference in the glucose concentration between the amyloglucosidase treated and untreated samples by tissue dry weight.

For determination of glycogen [³H]- and [¹⁴C]-specific activities, glycogen was precipitated in another aliquot (800 µl) of the alkaline extract with 1600 µl of 99.6% ethanol (-20 C, 2 h). After centrifugation (3000 rpm, +4 C, 30 min) (19, 24), the precipitate (ppt) was washed three times with 600 µl of ice-cold 66% ethanol and the supernatants from the ethanol washes were discarded. The washed ppt was dissolved in water, its pH was adjusted to 4.9 using 0.15 M sodium acetate buffer and an aliquot was taken for the determination of the glycogen concentration using the amyloglucosidase method (vide supra). The measurement of the glycogen concentration in the ethanol ppt was necessary because the recovery of glycogen in the ethanol precipitation procedure was less than 100% (data not shown). Another aliquot was counted for [³H]- and [¹⁴C]-radioactivity by dual channel counting (Rackbeta 1214, Wallac) and its glucose concentration was measured after amyloglucosidase treatment. Glycogen specific activity was calculated by dividing the [³H]- or [¹⁴C]-radioactivity (dpm/ml) by the glycogen concentration (µmol/ml) in the ethanol ppt. The amount of radioactivity incorporated into glycogen per dry muscle (dpm_gly) was calculated by multiplying glycogen specific activity (dpm/µmol) times the tissue glycogen concentration (mmol/kg dry).

Tissue preparation for ¹⁴C-DOG6P accumulation and GFA activity
For determination of the tissue concentration of ¹⁴C-DOG6P (dpm/ml) and the activities of GFA, frozen tissue specimens weighing 100–200 mg were homogenized (S25N-8G, Janke & Kunkel GmbH & Co., Staufen, Germany) for 15 sec in ice-cold 50 mM potassium phosphate buffer (pH 7.4) containing 2 mM dithiotreitol, 20 mM EDTA, 20 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 20 µg/ml p-aminobenzamidine, 70 µg/ml Na-p-tosyl-L-lysine chloromethyl ketone; and 170 µg/ml phenylmethylsulfonyl fluoride (27). The homogenates were centrifuged at 13,000 x g, and aliquots of the supernatant were taken for determination of the concentrations of protein and ¹⁴C-DOG6P and for measurement of GFA activity.

Tissue glucose uptake
For determination of the ¹⁴C-DOG6P concentration (dpm/ml), the homogenate was deproteinized with equal volume of ice-cold perchloric acid (1 mM, 10 min) and centrifuged (3000 G, 15 min). The supernatant was neutralized with 0.25 vol 2.2 M KHCO₃. An aliquot (500 µl) was passed through an anion-exchange (Ag 1-X8, acetate form, Bio-Rad, Hercules, CA) column, which was then washed with 2.5 ml of distilled water to elute nonanionic compounds such as glucose and glycogen. This eluate and another aliquot (500 µl, total radioactivity) of the neutralized sample were then counted for [¹⁴C]-radioactivity. The [¹⁴C]DOG6P concentration (dpm/ml) was calculated from the difference between the radioactivities in the total and neutral eluate radioactivities.

GFA activity
GFA activity was assayed as previously described (15). Briefly, after homogenization and centrifugation, 50 µl of the supernatant was incubated in a sodium phosphate buffered reaction mix (pH 7.4) containing EDTA and dithiotreitol and excess of F6P and glutamine for 45 min at 37 C in the presence and absence of excess of UDP-N-acetylglucosamine (UDP-GlcNAc), which is an allosteric inhibitor of GFA. After termination of the reaction with PCA, the sample was centrifuged, neutralized with KHCO₃, delipidated and derivatized with o-phtaldialdehyde (OPA) for 1 min before analysis of the GlcN6P concentration with a reverse-phase C₁₈ column (25 cm x 4.6 mm Spherisorb ODS, Phase Separations, Norwalk, CT). The mobile phase consisted of a one-step gradient made of 2.5% acetonitrile, 2.5% isopropanol in 30 mM sodium phosphate buffer and 12% acetonitrile, 12% isopropanol in 30 mM sodium phosphate buffer. Absorbance of the eluent was analyzed using a fluorescence detector and the peak area was integrated. OPA-derivatized standards were run separately to determine the retention time and to generate the standard curve to correlate area to activity. The correlation coefficient between the concentration of GlcN6P standards and the area under the GlcN6P peak was over 0.999. Internal and external standards were run in every assay. The recovery of samples spiked with GlcN6P before derivatization was 100%. Activity is expressed as pmols of GlcN6P formed per milligram protein per min. The coefficient of variation of GFA measurements in two pieces of rat muscle was <2%.

GlcN concentrations
Serum GlcN concentrations were assayed using the same sample preparation procedure and reverse-phase HPLC setup as in GlcN6P assay described above. The mobile phase consisted of a two-step gradient made of identical buffers as in GlcN6P assay. GlcN eluted with a retention time of 20.6 min. The correlation coefficient between the concentration of GlcN standards mixed in blank serum and the area under curve was 0.985.

Calculations
The rate of glucose appearance (R_a, µmol/kg·min) was calculated from the formula: R_a = (F/GSA)/W, where F denotes the isotope infusion rate (dpm/min), GSA the mean specific activity of glucose (dpm/µmol) in plasma, and W body weight (kg). The rate of liver glycogen synthesis (R_s; µmol/kg dry·min) was calculated from the formula R_s = [(dpm_gly/dt)]/GSA, where dt denotes the time period. The rate of whole body glycolysis was calculated from the increment in tritiated water radioactivity (dpm/ml·min) multiplied by total body water and divided by mean GSA (20). Tissue specific [¹⁴C]DOG uptake was calculated by dividing [¹⁴C]DOG6P and [¹⁴C]glycogen radioactivity (dpm/mg tissue) per mg protein with the total plasma area under the curve of [¹⁴C]DOG specific activity (dpm·min/µmol plasma glucose). This formula is similar to that validated by Hom et al. (28) and Jenkins et al. (29), with the exception that the amount of ¹⁴C-radioactivity incorporated into glycogen from [¹⁴C]DOG is also taken into account. This is necessary since DOG6P is incorporated into glycogen (30, 31) under insulin-stimulated conditions (32).

Statistical analysis
Data between the study groups were analyzed using the unpaired Student’s t test. Simple correlation’s between selected study variables were calculated using Pearson’s correlation coefficient for variables that were not normally distributed. All statistical calculations were made using the BMDP statistical software (BMDP Statistical Software, Los Angeles, CA). All data are expressed as means ± SE.

	Results

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Whole body insulin sensitivity and glucose kinetics
Fasting plasma glucose concentrations were similar between the groups (data not shown). During hyperinsulinemia, serum insulin concentrations averaged 1657 ± 253 pmol/liter in INS and 1290 ± 314 pmol/liter (NS) in GLCN rats, and plasma glucose concentrations 5.6 ± 0.1 mmol/liter and 5.6 ± 0.1 mmol/liter, respectively. Plasma glucose averaged 5.7 ± 0.1 mmol/liter in the BAS rats (NS vs. INS and GLCN rats). Plasma GlcN concentrations averaged 0.81 ± 0.10 mmol/liter in GLCN rats and were undetectable in BAS and INS rats.

Figure 1 shows the rate of glucose uptake as a function of time, as determined from the glucose infusion rate necessary to maintain normoglycemia in the INS and GLCN rats. The rate of glucose uptake was similar in INS (210 ± 7 µmol/kg·min) and GLCN rats (170 ± 10 µmol/kg·min) between 60–120 min, but during the last hour of the study the rate of glucose uptake was 49% lower in the GLCN (208 ± 8 µmol/kg·min) than the INS (107 ± 11 µmol/kg·min, P < 0.001) rats. During the entire study (0–360 min), the rate of glucose uptake was 30% lower in the GLCN (168 ± 9 µmol/kg·min) than the INS (239 ± 8 µmol/kg·min), P < 0.001) rats. The rate of glucose turnover in the BAS rats averaged 49 ± 5 µmol/kg·min. The rate of glucose turnover (R_t) during 60–360 min of hyperinsulinemia measured with isotopic dilution method (\[3-³H\]glucose) was significantly lower in the GLCN (151 ± 7 µmol/kg·min) than the INS (192 ± 7 µmol/kg·min, P < 0.05) rats. The greatest difference between INS and GLCN rats in R_t was observed during the last hour of the study (300–360 min), when R_t averaged 133 ± 7 µmol/kg·min in the GLCN and 194 ± 8 µmol/kg·min in the INS rats (P < 0.001). R_t:s at 60–120 min were comparable between GLCN (169 ± 7 µmol/kg·min) and INS (186 ± 6 µmol/kg·min, NS) rats. These isotopically measured rates of glucose turnover were not different from the glucose infusion rate needed to maintain euglycemia confirming that hepatic glucose production was completely suppressed in both GLCN and INS rats. Rates of whole body glycolysis (60–360 min) were similar in GLCN (74 ± 5 µmol/kg·min) and INS (74 ± 5 µmol/kg·min, NS) rats.

View larger version (18K): [in this window] [in a new window]   Figure 1. The rate of whole body glucose uptake in INS and GLCN rats as a function of time. The hatched line depicts the basal rate of glucose turnover measured in BAS rats. *, P < 0.05; **, P < 0.01; ***, P < 0.001, INS vs. GLCN.

View larger version (34K): [in this window] [in a new window]   Figure 2. Tissue glucose uptake rates measured with 2-deoxyglucose in the submandibular gland (SG), epididymal fat (Fat), in soleus, abdominis (ABD), and gastrocnemius (GAS) muscles and in the heart. The rate of glycogen synthesis measured using [3-3H]glucose in the liver in BAS (white bars), INS (gray bars) and GLCN (hatched bars) is shown on the right. ###, P < 0.001 BAS vs. INS; *, P < 0.05; **, P < 0.01; ***, P < 0.001 GLCN vs. INS; P < 0.01 soleus vs. abdominis.

Glycogen concentrations and rates of glycogen synthesis
Tissue glycogen concentrations were significantly higher in INS than in BAS rats in soleus, abdominis and gastrocnemius (Table 1). Infusion of GlcN abolished glycogen synthesis in the liver (228 ± 40 vs. 1 ± 5 µmol/kg dry·min, Fig. 2) and significantly decreased glycogen content in abdominis muscle (Table 1). In contrast, the glycogen content increased significantly in the heart and was unaltered in soleus and gastrocnemius muscles (Table 1).

View larger version (25K): [in this window] [in a new window]   Figure 3. Basal GFA activities (top panel), GlcN induced absolute (middle panel), and percent (bottom panel) decreases in insulin stimulated 2-deoxyglucose uptake in the submandibular gland, epididymal fat, soleus, abdominis, and gastrocnemius muscles and in the heart, and the rate of glycogen synthesis in the liver. Legends as in Fig. 2. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

	Discussion

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The present data demonstrate first, that induction of insulin resistance by infusion of GlcN in vivo is not restricted to abdominis muscle but is also observed in other tissues such as the heart, fat, the liver, submandibular gland, soleus, and hindlimb muscles. Second, we found that changes in glycogen concentrations and rates of glycogen synthesis induced by GlcN showed considerable variation ranging from complete inhibition of glycogen synthesis in the liver to an increase in glycogen concentrations in the heart. Finally, we found that 6 h of hyperinsulinemia did not alter GFA activities in skeletal muscle or adipose tissue but did increase GFA activity in the heart and in the submandibular gland.

The dose of GlcN and the 30% decrease in whole body glucose uptake induced by GlcN in the present study are similar to those described by Rossetti et al (30% decrease over 5 h) (9). In keeping with both the latter data (9) and those of Giaccari et al (8), the decrease in whole body glucose uptake was associated with a 31% decrease in glucose uptake in abdominis muscle. Abdominis muscle consists mostly of glycolytic, white or type 2a fibers (33) and has lower rates of insulin-sensitive glucose uptake than muscles such as the soleus which consists of insulin-sensitive, type 1 fibers (34). These characteristics were confirmed in the present study as both the absolute rates of glucose uptake (6.4 ± 0.7 vs. 2.2 ± 0.3 nmol/mg protein·min, soleus vs. abdominis muscle, P < 0.05) and the insulin induced increases in glucose uptake above basal (4.6 ± 0.5 vs. 2.1 ± 0.3 nmol/mg protein·min, respectively, P < 0.05) were significantly higher in soleus than abdominis muscles. Insulin-stimulated rates of glucose uptake were also inhibited to a significantly greater extent in soleus (by 2.4 nmol/mg protein·min) than in abdominis (by 0.7 nmol/mg protein·min) muscle, suggesting that the sensitivity of inhibition of glucose uptake by the hexosamine pathway depends on muscle fiber composition or insulin sensitivity.

In addition to skeletal muscle, we observed a significant decrease in glucose uptake by GlcN in adipose tissue, the heart, liver and the submandibular gland. Both the absolute rates of insulin stimulated glucose uptake, expressed per mg of protein, and the percent decrease by GlcN were comparable in the submandibular gland and in hindlimb muscle (Figs. 2 and 3). These data imply that basal GFA activity, which was 10-fold higher in the submandibular gland than in hindlimb muscle, does not determine the extent to which a tissue is susceptible to inhibition of glucose uptake via activation of the hexosamine pathway. The assumption that the proportion of glucose metabolized via the hexosamine pathway is determined by GFA is supported by several observations. First, GFA is the rate-limiting enzyme of the pathway (4). Second, accumulation of hexosamine metabolites seems to parallel GFA activity, as a 2-fold overexpression of the enzyme in transgenic mice (5) doubles the concentration of UDP-N-acetyl-hexosamines. Third, UDP-N-acetyl-hexosamine concentrations parallel GFA activities between tissues (7). The dissociation between the extent of insulin resistance of glucose uptake vs. GFA activity of a particular tissue could be due to tissue specific differences in the intracellular channeling of products of the hexosamine pathway, particularly UDP-GlcNAc to pathways mediating insulin resistance and to those not involved in glucose uptake regulation. The former could involve changes in O-linked glycosylation of intracellular proteins, which is known to regulate the function of several proteins and is often reciprocal to protein phosphorylation (35). The pathways not involved in regulation of insulin sensitivity would then be those utilizing UDP-GlcNAc for synthesis of glycolipids and extracellular N-linked glycoproteins (36, 37). Consistent with the idea that O-linked glycosylation might mediate insulin resistance, the submandibular gland uses a major proportion of UDP-GlcNAc for production of extracellular glycoproteins and mucins (38), whereas O-linked glycosylation predominates in skeletal muscle (39). Also, we have recently demonstrated that the activity of specific UDP-GlcNAc transferase, which catalyzes final O-linked attachment of UDP-GlcNAc to serine and threonine residues on intracellular proteins, is equally active in the submandibular gland and in gastrocnemius muscles although GFA activity was markedly higher in the submandibular gland than in gastrocnemius muscle (13).

The absolute GlcN-induced decrease in whole body glucose uptake became significant after 2 h and reached a maximum of 101 µmol/kg·min between 5 and 6 h (Fig. 1). Because the magnitude of the maximal decrease in glucose uptake was more than three times greater than the infusion rate of GlcN (30 µmol/kg·min), simple competition between glucose and GlcN cannot explain the GlcN-induced insulin resistance. These data are consistent with those of Rossetti et al. (9). In addition, the time delay in the ability of GlcN to induce insulin resistance and the modest inhibition of muscle hexokinase by GlcN (9) also argues against simple competition as the mechanism underlying GlcN induced insulin resistance. Because skeletal muscles consume the majority of glucose under normoglycemic hyperinsulinemic conditions in the rat (40), simple competition is also unlikely to explain insulin resistance in skeletal muscle. On the other hand, this may not apply to tissues such as the liver where glucose phosphorylation by glucokinase is rate-limiting for glucose uptake and where intracellular glucose concentrations equal extracellular glucose concentrations (41). Under such conditions in the liver, inhibition of glucokinase by GlcN may not be trivial. Clearly, further studies are needed to determine the exact cause(s) for abolition of glycogen synthesis by GlcN in the liver.

Consistent with the data of Rossetti et al. (9) and those of Giaccari et al. (8), we found a significant decrease in the rate of glycogen synthesis in abdominal muscle. On the other hand, the glycogen concentrations in soleus and hindlimb muscles were unchanged by GlcN. The latter data are in keeping with those of Fürnsinn et al. (17), who observed diminished total glucose uptake in the face of inhibition of glycolysis and increased incorporation of glucose into glycogen in the presence of insulin and GlcN compared to insulin alone in the isolated soleus muscle in vitro. Other studies have shown GlcN to increase the fractional velocity and total activity of glycogen synthase activity in Rat-1 fibroblasts overexpressing the human insulin receptor (10), and an increase in basal glycogen synthase activity in GFA-transfected fibroblasts (6). The apparent increase in glycogen synthase activity coupled with increased channeling of glucose to glycogen in the heart, and two of the three skeletal muscle examined, implies that GlcN does not perfectly mimic insulin resistance caused by chronic hyperglycemia, which has invariably been associated with diminished rates of glycogen synthesis in both rats (20) and patients with IDDM (42) and NIDDM (15). One possibility to explain the increased rates of glycogen synthesis is that GlcN induces a large increase in GlcN6P concentrations, which stimulate glycogen synthase activity and consequently glucose incorporation into glycogen (17).

The tissue distribution of GFA activities found in the BAS rats in the present study contrasts earlier failure of Kaufman et al. (12) to detect significant GFA activity in heart or skeletal muscle using a colorimetric assay for detection of multiple acetylated hexosamines but is consistent with more recent data obtained using a sensitive and specific HPLC method for measurement of GFA activity (7, 13, 15, 16). Regarding the effect of insulin on GFA activity, we found no change in skeletal muscle GFA activity during 6 h of hyperinsulinemia in rats. This result is in keeping with our previous data showing no change in GFA activity in human skeletal muscle in normal subjects or patients with NIDDM during 4 h of hyperinsulinemia (15), or in control, insulin-deficient diabetic or insulin treated rats 1 or 2 h after a glucose injection (7). Long-term exposure of cultured human skeletal muscle cells to hyperinsulinemia (16), hyperglycemia (16) does, however, increase GFA activity (16), and an increase in GFA activity also characterizes chronically hyperglycemic patients with NIDDM (15). Stimulation of GFA activity in the heart and in the submandibular gland by insulin has not been previously reported but is in line with the data demonstrating insulin induced increases in skeletal muscle GFA activity. These data, together with the lack of an effect of increased GlcN concentrations on GFA activity in adipose tissue in the present study, differs from the data by Marshall et al., who found decreases in GFA activity within a few hours both by low concentrations GlcN, and by the combination of glucose, insulin and glutamine in primary cultures of rat adipocytes (14).

In conclusion, the present data demonstrate that infusion of GlcN to normal rats induces insulin resistance in several insulin sensitive tissues including fast- and slow-twitch skeletal muscles, the heart, liver, adipose tissue, and the submandibular gland. The magnitude of insulin resistance exhibited tissue specificity and was greatest in the liver and lowest in the fast-twitch glycolytic abdominis muscle. These data raise the possibility that overactivity of the hexosamine pathway may contribute to glucose toxicity not only in skeletal muscle but also in the heart and in the liver.

	Footnotes

 
¹ Supported by grants from the Academy of Finland (H.Y., A.V.) and the Sigrid Juselius Foundation (H.Y., A.V.).

Received November 8, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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