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Muscle Uridine Diphosphate-Hexosamines Do Not Decrease Despite Correction of Hyperglycemia-Induced Insulin Resistance in Type 2 Diabetes

Divisions of Endocrinology (M.-J.J.P., A.R.H.) and General Internal Medicine (M.-J.J.P., C.J.T., J.A.L.), Department of Medicine, and Departments of Chemical Endocrinology (P.N.S., A.J.O., C.G.J.S.) and Neurology (B.G.v.E.), Laboratory for Pediatrics and Neurology (J.G.d.J.), University Medical Center, 6500 HB Nijmegen, The Netherlands

Address all correspondence and requests for reprints to: Marie-Jose Pouwels, M.D., Division of General Internal Medicine, Medisch Spectrum Twente, P.O. Box 50000, 7500 KA Enschede, The Netherlands. E-mail: m.j.pouwels{at}club.tip.nl.

Abstract

Animal studies suggest that overactivity of the hexosamine pathway, resulting in increased UDP-hexosamines [UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc)] is an important mechanism by which hyperglycemia causes insulin resistance. This study was performed to test this hypothesis in patients with type 2 diabetes mellitus (DM).

Eight obese patients with uncontrolled DM type 2 and severe insulin resistance were treated with iv insulin for 28 ± 6 d aimed at euglycemia. Before and after iv insulin treatment, insulin sensitivity was measured using a hyperinsulinemic euglycemic clamp, and a muscle biopsy was taken for measurement of UDP-GlcNAc, UDP-GalNAc, UDP-glucose, and UDP-galactose levels. Also, isoelectric focusing patterns of serum transferrin and the urinary excretion of glycosaminoglycans as measures of final products of the hexosamine pathway were examined.

After euglycemia, insulin resistance improved, as demonstrated by an increase in the glucose infusion rate during the clamp from 12.7 ± 5.6 to 22.4 ± 8.8 µmol/kg·min (P < 0.0005) and a decrease in insulin requirement from 1.7 ± 0.9 to 1.1 ± 0.6 U/kg·d (P < 0.005), whereas metabolic control improved. Surprisingly, both UDP-GlcNAc, from 8.81 ± 1.21 to 12.31 ± 2.52 nmol/g tissue (P < 0.005), and UDP-GalNAc concentrations, from 4.49 ± 0.85 to 5.89 ± 1.55 nmol/g tissue (P < 0.05) increased. Isoelectric focusing patterns of serum transferrin and excretion of glycosaminoglycans were similar before and after euglycemia.

In conclusion, after amelioration of hyperglycemia- induced insulin resistance, UDP-hexosamines increased in skeletal muscle of patients with type 2 DM. These results do not support the hypothesis that accumulation of products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance.

CHRONIC HYPERGLYCEMIA is not only a consequence of type 2 diabetes mellitus (DM), but also aggravates the already impaired secretion and action of insulin in these patients, thereby self-perpetuating the diabetic state. These adverse metabolic consequences of hyperglycemia have been conceptualized as glucose toxicity (1).

Animal studies suggest that accumulation of products of the hexosamine biosynthetic pathway plays a major role in hyperglycemia-induced insulin resistance (2, 3, 4, 5). Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme that regulates the flux through this pathway by catalyzing the formation of glucosamine-6-phosphate (GlcN-6-P) from fructose-6-phosphate and glutamine (6, 7, 8). GlcN-6-P is then converted to UDP-hexosamines, i.e. UDP-N-acetylglucosamine (UDP- GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc). These products serve as substrates in the biosynthesis of glycoproteins, proteoglycans, and glycolipids (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. The hexosamine pathway in skeletal muscle. After glucose uptake into the cell by glucose transporters, such as GLUT-4, glucose is phosphorylated to glucose-6-phosphate (Glucose-6-P). Thereafter, glucose is used by the major pathways, glycogen synthesis and glycolysis. Normally 2–3% of intracellular glucose enters the hexosamine pathway. GFAT is the key enzyme that regulates the flux through this pathway and catalyzes the formation of GlcN-6-P from fructose-6-phosphate and glutamine. GlcN-6-P is then converted to UDP-GlcNAc and UDP-GalNAc and finally to glycoproteins, glycolipids, and proteoglycans. Glucosamine is also transported into the cell by GLUT4 and then directly enters the hexosamine pathway, bypassing GFAT.

To date, human studies concerning the role of the hexosamine pathway in hyperglycemia-induced insulin resistance are scarce and restricted to measurements of GFAT enzyme activity. Both positive (13) and negative (14) correlations between GFAT activity in human muscle tissue from patients with type 2 DM and glucose disposal rate have been reported.

The present study was designed to test the hypothesis that chronic hyperglycemia-induced insulin resistance is caused by accumulation of products of the hexosamine pathway in patients with type 2 DM. In this prospective intervention study, metabolic products of the hexosamine biosynthetic pathway were measured in muscle tissue of severely insulin-resistant patients with type 2 DM before and after intensive treatment with iv insulin. We also studied whether this intensive iv insulin treatment resulted in a change in glycosylation of glycoproteins and synthesis of glycosaminoglycans (GAGs; degradation products of proteoglycans) representing the final products of the hexosamine pathway. We hypothesized that if accumulation of products of the hexosamine biosynthetic pathway plays a major role in hyperglycemia-induced insulin resistance, the levels of UDP-hexosamines in muscle tissue should decrease, and glycosylation of glycoproteins and excretion of GAGs might decrease, after correction of hyperglycemia.

Subjects and Methods

Subjects

The study group consisted of eight insulin-treated patients (female/male ratio, 6:2; mean ± SD age, 53 ± 13 yr) with type 2 DM, who were markedly obese (body mass index, 38 ± 5.8 kg/m²; waist/hip ratio, 0.97 ± 0.08) and severely insulin resistant [hemoglobin A_1c (HbA_1c), 12.0 ± 1.7%; sc insulin dose, 1.92 ± 0.66 U/kg·d]. Metabolic control, weight, and insulin treatment (four sc injections per day) were stable in all patients for at least 3 months before the study. None of the patients had clinical or biochemical evidence of any other endocrine disease. All patients gave informed consent. The hospital ethics committee approved the experimental protocol.

Protocol

Patients were admitted into the hospital for iv insulin treatment to achieve euglycemia. Before and after reaching euglycemia, patients underwent a muscle biopsy and a clamp procedure. In addition, blood and urine samples were obtained.

On the first day after admission a muscle biopsy was taken, followed by a hyperinsulinemic, euglycemic clamp study on the first (two patients), the second (five patients), or the third (one patient) day. Thereafter patients were treated continuously with iv insulin (Actrapid, Novo-Nordisk, Copenhagen, Denmark) to achieve a period of euglycemia for at least 10 d. In the hospital, patients were on a diet consisting of 1500 kcal/24 h according to standard nutritional recommendations. Capillary blood glucose (CBG) was measured five times daily (fasting, prelunch, presupper, at bedtime, and at 0300 h). Euglycemia was considered to be reached when all CBG measurements over 24 h were between 4.0–6.5 mmol/liter. The insulin dose was individually titrated and increased until euglycemic values were obtained. Subsequently, the insulin dose required for maintaining euglycemia gradually decreased.

The second muscle biopsy was taken after iv insulin treatment for 27 ± 4 d (range, 21–33 d) when patients were euglycemic for 16 ± 3 d (range, 11–21 d). The second hyperinsulinemic, euglycemic clamp was performed on the same day as the second muscle biopsy (one patient) or 1 (three patients) or 2 d later (4 patients) when patients were euglycemic for 17 ± 4 d (range, 12–22 d). On the days of the clamps fasting levels of glucose, HbA_1c, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides, and nonesterified fatty acids (NEFA) were measured. In addition isoelectric focusing of serum transferrins was performed.

Twenty-four-hour urine was collected for measurement of glycosaminoglycan excretion 2 ± 2 d (range, 0–4 d) after admission and 29 ± 7 d (range, 21–41 d) after admission, after an euglycemic period of 19 ± 3 d (range, 16–24 d).

Muscle biopsies

Patients were studied while fasting. CBG was measured before biopsy. Muscle biopsies (30–100 mg) were taken for measurement of UDP-glucose, UDP-galactose, UDP-GlcNAc, and UDP-GalNAc levels from vastus lateralis muscle under local anesthesia using a Bergström needle. The muscle tissues were immediately frozen in liquid nitrogen and stored at -80 C until analysis.

Hyperinsulinemic, euglycemic clamp

Insulin sensitivity was measured using the hyperinsulinemic clamp technique (15). Insulin (Actrapid, Novo-Nordisk; diluted in a solution of 0.9% NaCl with 4% albumin to a concentration of 2 U/ml) was infused in a dose of 120 mU/m² ·min (720 pmol/m² ·min) for approximately 4 h. Patients were clamped at a glucose level of 5.5 mmol/liter, which was maintained by a variable infusion of 20% glucose solution, adjusted by plasma glucose measurements performed every 5 min. Plasma glucose levels during the clamp procedures were stable in all patients during the last hour. During the steady state the glucose infusion rate was measured and expressed as micromoles per kilograms per minute.

Laboratory assays

Plasma glucose was measured using the glucose oxidation method (Glucose Analyzer II, Beckman, Palo Alto, CA). Plasma insulin was determined using an in-house RIA (interassay coefficient of variation, 6%). Hemoglobin A_1c (reference range, 4.8–6.2%) was determined with an HPLC technique (Bio-Rad Laboratories, Inc., Richmond, CA). The concentrations of NEFA were analyzed using an enzymatic method (ACS-ACOD, NEFA C-kit, WACO Chemicals, Neuss, Germany). Triglycerides (Triglycerides GPO-PAPmethod), were determined on a Hitachi 747 autoanalyzer (Roche Molecular Biochemicals, Indianapolis, IN).

UDP-hexosamines, i.e. UDP-GlcNAc and UDP-GalNAc, and UDP-hexoses, i.e. UDP-glucose and UDP-galactose, were measured using an HPLC-based assay suitable for analyzing small samples of human muscle tissue, as previously described (16). For UDP-GlcNAc, intra- and interassay coefficients of variation (CV) were less than 6%. For UDP-GalNAc, the intraassay CV was 5.4%, and the interassay CV was less than 13%. For UDP-glucose and UDP-galactose, the intraassay CV were less than 6.5%, and the interassay CV were less than 7%. The limit of detection for all metabolites (UDP-glucose, UDP-galactose, UDP- GlcNAc, and UDP-GalNAc) was 0.6 nmol/g tissue (16).

Isoelectric focusing of serum transferrins was performed as previously described (17). Patterns were investigated by visual inspection. Usually transferrin contains four residues of neuraminic acids. This might differ from zero to six residues, reflecting the extent of glycosylation of the transferrin molecules, resulting in different isoelectric focusing patterns.

GAG excretion was measured using the dimethylmethylene blue (DMB) assay in six patients and the DMB-Tris assay in eight patients as previously described (18, 19). The DMB assay is more specific for GAGs than the DMB-Tris assay, but is disturbed by excretion of protein (micro- or macroalbuminuria) (19). The DMB-Tris assay is less specific for GAGs compared with the DMB assay, because of a higher background measurement. The DMB-Tris assay is therefore characterized by higher values, which is also reflected in higher reference values (19). The presence of microalbuminuria in one patient and macroalbuminuria in another patient made it impossible to use the DMB assay in these two patients (19). Because of these characteristics, excretion of GAGs was measured with both assays.

Statistical analysis

Statistical analyses of differences were evaluated by t test (two-tailed, paired). Correlation coefficients were assessed using Spearman’s rank method (SPSS package, SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant. Results are given as the mean ± SD unless otherwise stated.

Results

Metabolic effects of intervention (iv insulin treatment)

Euglycemia was reached after iv insulin treatment for 12 ± 6 d (range, 6–24 d; Fig. 2). Intravenous insulin treatment improved insulin sensitivity, as reflected by a decrease in iv insulin requirement to achieve and maintain euglycemia from 1.7 ± 0.9 to 1.1 ± 0.6 U/kg·d (P < 0.005). In addition, the glucose infusion rate measured during the clamp studies increased (from 12.7 ± 5.6 to 22.4 ± 8.8 µmol/kg·min; P < 0.0005). Baseline and steady state (240 min) plasma insulin concentrations during both clamps were similar. Metabolic control improved; HbA_1c decreased (from 12.0 ± 1.7% to 8.6 ± 1.1%; P < 0.0001), triglyceride levels tended to decrease (from 4.34 ± 3.32 to 1.91 ± 0.69 mmol/liter; P = 0.07). NEFA concentrations were similar before and after treatment (1.49 ± 0.81 vs. 1.04 ± 0.59 mmol/liter; P = 0.35). Capillary blood glucose levels, measured before the muscle biopsy, were lower before the second biopsy than before the first biopsy (6.0 ± 1.2 vs. 13.8 ± 4.1 mmol/liter; P < 0.01).

View larger version (11K): [in this window] [in a new window]   Figure 2. Average 24-h glucose values during admission (mean ± SE). Each day the average of three premeal, one bedtime, and one nighttime (0300 h) glucose determinations using capillary blood glucose measurements was calculated in each patient.

After iv insulin treatment, UDP-GlcNAc and UDP-GalNAc concentrations were significantly higher than before treatment (Fig. 3). UDP-GlcNAc increased from 8.81 ± 1.21 to 12.31 ± 2.52 nmol/g tissue (P < 0.005), and UDP-GalNAc increased from 4.49 ± 0.85 to 5.89 ± 1.55 nmol/g tissue (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Figure 3. UDP-GlcNAc (top) and (bottom) UDP-GalNAc concentrations in muscle tissue before and after reaching euglycemia: individual responses. Both UDP-GlcNAc and UDP-GalNAc concentrations increased significantly (P < 0.005 for UDP-GlcNAc and P < 0.05 for UDP-GalNAc).

No statistically significant correlations between muscle concentrations of UDP-GlcNAc and UDP-GalNAc and glucose infusion rate were found either before or after euglycemia (data not shown).

Isoelectric focusing patterns of transferrin

The glycosylation of transferrin was not different before and after reaching euglycemia (Fig. 4).

View larger version (81K): [in this window] [in a new window]   Figure 4. Isoelectric focusing pattern of serum transferrins in eight patients. A, Before euglycemia; B, after euglycemia. Fraction 0–5 corresponds to sialotransferrins containing zero to five residues of neuraminic acids, reflecting the extent of glycosylation of the transferrin molecules. After euglycemia, the patterns of transferrin were unchanged (usually transferrin contains four residues of neuraminic acids).

Excretion of GAGs, measured by DMB, either expressed as milligrams per millimoles of creatinine or milligrams per 24 h, was not significantly different before and after reaching euglycemia (1.39 ± 0.25 before vs. 1.43 ± 0.3 mg/mmol creatinine after euglycemia and 21.7 ± 8.5 before vs. 18.5 ± 8.5 mg/24 h after euglycemia, respectively; n = 6; two patients were excluded because of albuminuria). Similar results were obtained when excretion of GAGs were measured by DMB-Tris assay (3.0 ± 0.9 before vs. 3.5 ± 0.7 mg/mmol creatinine after euglycemia; 46.4 ± 25.7 before vs. 42.0 ± 15.7 mg/24 h after euglycemia, respectively; n = 8).

Discussion

The results of this study provide strong evidence against the hypothesis that accumulation of metabolic products of the hexosamine pathway plays a major role in the mechanism by which hyperglycemia induces insulin resistance in humans.

In our group of severely insulin-resistant subjects who were in bad metabolic control, a 4-wk period of iv insulin treatment dramatically improved metabolic control and insulin sensitivity, as reflected by a decreased insulin requirement to maintain euglycemia and an increase in glucose infusion rate during the clamp. Despite this amelioration of hyperglycemia-associated insulin resistance, muscular levels of UDP-GlcNAc and UDP-GalNAc, key products of the hexosamine pathway, did not decrease, but, surprisingly, even increased. In addition, isoelectric focusing patterns of transferrin and urinary excretion of GAGs as measurements of final products of the hexosamine pathway did not change. Together, these findings make it highly unlikely that accumulation of metabolic products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance in this group of patients.

Before and after euglycemia we measured insulin sensitivity using a hyperinsulinemic euglycemic clamp. It is well known that at high insulin levels the endogenous hepatic glucose production is almost completely suppressed (20, 21). We conclude from the fact that glucose infusion rate increased that peripheral insulin sensitivity improved. Although not formally excluded, we believe that it is very unlikely that the increase in the steady state glucose infusion rate is explained by a change in hepatic glucose production.

In the literature, a fairly large body of evidence from in vitro and animal experiments suggests that increased flux into the hexosamine pathway results in insulin resistance. For example, in Rat-1 fibroblasts that overexpress GFAT, both elevated UDP-GlcNAc levels and insulin resistance were found (22). Also, several animal studies revealed a positive correlation between activity of the hexosamine biosynthetic pathway, resulting in increased UDP-GlcNAc and UDP-GalNAc levels, and insulin resistance (3, 5, 9, 10, 23). Especially Rossetti and co-workers have clearly demonstrated a relation between overactivity of the hexosamine pathway induced by glucosamine infusion (3, 5, 10) or induced by hyperglycemia (5) or fat (5), and insulin resistance in animal studies. The discrepancy between the results obtained by Rossetti and our results can be explained by differences in species, the presence or absence of diabetes, and the experimental design, including the duration of hyperglycemia.

Whereas induction of insulin resistance by activation of the hexosamine pathway is fairly well documented, only a few animal studies have actually investigated the relationship between hyperglycemia and the activity of the hexosamine pathway. In a study in rats, UDP-hexosamines increased and UDP-hexoses decreased in rats with streptozotocin-induced diabetes (4). In contrast with our patients who are characterized by prolonged extensive hyperinsulinemia, these diabetic rats were hypoinsulinemic and had diabetes for a relatively short period. In another study (3), normal rats, pancreatectomized (hypoinsulinemic) rats, and pancreatectomized rats treated with phlorizin (which ameliorates glucose-induced insulin resistance) (24) were studied under baseline conditions and after glucosamine infusion. This study (3) found similar baseline levels of UDP-hexosamines in the three groups. Apparently UDP-hexosamine levels were not increased in the diabetic rats compared with the normal rats and the diabetic rats treated with phlorizin. These findings argue against involvement of the hexosamine pathway in hyperglycemia-induced insulin resistance and are in line with our results.

Human studies of the relationship between the hexosamine pathway and insulin resistance are scarce (13, 14, 25, 26). We and others have reported that in normal volunteers, short-term glucosamine infusion, which should result in increased flux through the hexosamine pathway, did not affect insulin sensitivity (25, 26). In one study using human skeletal muscle cultures of patients with type 2 DM, a positive correlation between GFAT activity and the glucose disposal rate was found (13). In contrast, in another study a negative correlation between GFAT activity in the muscle of patients with type 2 DM and the glucose disposal rate was reported (14). In neither of these two studies were concentrations of UDP-hexosamine and UDP-hexose metabolites in skeletal muscle measured (13, 14). To our knowledge, the present study is the first that quantifies these products of the hexosamine pathway in skeletal muscle in diabetic patients.

UDP-hexose concentrations were not detectable in our patients either before or after euglycemia. As UDP-glucose is the obligate substrate for glycogen synthase, the most straightforward explanation is that a decreased concentration of UDP-glucose mirrors the decreased glucose uptake in the cell and the subsequent decreased flux into glycogenesis. In essence, this represents the features of insulin resistance (27). Even after iv insulin treatment and reversion of the hyperglycemia-induced insulin resistance, UDP-hexoses remained undetectably low. Previously we found that UDP-glucose in muscle tissue (gluteus maximus) of type 2 diabetic patients was lower than that in muscle tissue of control subjects (5.81 vs. 13.9 nmol/g tissue) (16). The levels in that study were all well above the limit of detection. We conclude that the undetectably low UDP-hexose levels reflect the fact that our patients remained insulin resistant despite improvement after correction of chronic hyperglycemia.

As UDP-hexosamine metabolites are the precursors for the formation of glycoproteins, proteoglycans, and glycolipids (2), final products of the hexosamine pathway, we also investigated whether glycosylation of glycoproteins and synthesis of GAGs changed after treatment. Isoelectric focusing patterns of transferrin and urinary excretion of GAGs were similar before and after euglycemia. The discrepancy between the change in UDP-GlcNAc and the lack of change in GAGs may be explained by the fact that transferrin and GAGs were measured in blood and urine, respectively, where changes may be smaller than in muscle tissue. An alternative explanation is that the synthesis is regulated in such a way that increased concentrations of hexosamine precursors do not automatically result in increased synthesis of end products of the hexosamine pathway. As this study was performed in eight patients, the possibility of a type 2 error also cannot be disregarded.

Our study has some limitations. As in other studies (3, 5, 9, 10, 23), we measured overall muscle UDP-GlcNAc and UDP-GalNAc. Therefore, although unlikely, we cannot exclude the possibility that UDP-GlcNAc and UDP-GalNAc levels might have decreased in a specific muscle compartment despite increased overall muscle UDP-hexosamines. Furthermore, for ethical reasons we could not study a diabetic control group (diabetic patients who were admitted to the hospital, but who did not receive insulin infusion) to determine the unlikely possibility that hexosamine concentrations may change over time without intervention.

In summary, the present intervention study in obese patients with type 2 DM, insulin resistance, and chronic hyperglycemia provides strong evidence against the hypothesis that accumulation of products of the hexosamine pathway plays a major role in hyperglycemia-induced insulin resistance, at least in this category of patients.

Acknowledgments

Mrs. D. Jankowiak (Department of Chemical Endocrinology), Ms. K. Huyben, and Ms. R. Liebrand-van Sambeek (Laboratory for Pediatrics and Neurology) are acknowledged for their technical assistance.

Footnotes

This work was supported by a grant from the Dutch Diabetes Foundation.

Abbreviations: CBG, Capillary blood glucose; CV, coefficient of variation; DM, diabetes mellitus; DMB, dimethylmethylene blue; GAG, glycosaminoglycan; GFAT, glutamine fructose-6-phosphate amidotransferase; GlcN-6-P, glucosamine-6-phosphate; HbA_1c, hemoglobin A_1c; NEFA, nonesterified fatty acids; UPD-GlcNAc, UDP-N-acetylglucosamine; UDP-GalNAc, UDP-N-acetylgalactosamine.

Received March 20, 2002.

Accepted August 5, 2002.

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