1 Division of Endocrinology, Enhanced glucose flux via the hexosamine
biosynthesis pathway (HNSP) has been implicated in insulin resistance.
We measured L-glutamine:D-fructose-6-phosphate
amidotransferase activity (GFAT, a rate-limiting enzyme)
and concentrations of UDP-N-acetyl
hexosamines (UDP-HexNAc, major products of HNSP) in muscle and liver of
growth hormone (GH)-deficient male dwarf (dw) rats. All parameters
measured, except body weight, were similar in 5-wk-old control and dw
rats. Muscle GFAT activity declined progressively with age in controls and dw rats but was consistently 30-60% lower in 8- to 14-wk-old dw rats vs. age-matched controls; UDP-HexNAc concentrations in muscle
were concomitantly 30% lower in dw rats vs. controls
(P < 0.01). Concentrations of
UDP-hexoses, GDP-mannose, and UDP in muscle were similar in control and
dw rats. Muscle HNSP activity was similarly diminished in fed and
fasted dw rats. In liver, only a small difference in GFAT activity was
evident between controls and dw rats, and no differences in UDP-HexNAc
concentrations were observed. Treatment with recombinant human GH
(rhGH) for 5 days restored UDP-HexNAc to control levels in dw muscles
(P < 0.01) and partially restored
GFAT activity. Insulin-like growth factor I treatment was ineffective.
We conclude that GH participates in HNSP regulation in muscle.
L-glutamine:D-fructose-6-phosphate
amidotransferase activity in muscle and liver; UDP-N-acetyl hexosamine concentration
in muscle and liver; regulation of hexosamine biosynthesis by growth
hormone in muscle; growth hormone-induced insulin resistance
INSULIN RESISTANCE is a hallmark of
non-insulin-dependent diabetes mellitus and is also associated with
uncontrolled insulinopenic diabetes in humans and rodents. In the
former condition, insulin resistance is thought to be genetically
determined, whereas in the latter, it is reversed by normalizing
circulating glucose concentrations and attributed to "glucose
toxicity" (10). On the basis of studies in adipocytes in primary
culture, Marshall et al. (19) proposed in 1991 that increased flux of
glucose via the hexosamine synthesis pathway (HNSP) may contribute to glucose-induced insulin resistance of glucose transport. The
rate-limiting enzyme for glucose flux via HNSP is
L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT), which catalyzes the conversion of fructose 6-phosphate and glutamine to glucosamine 6-phosphate
(GlcN-6-P) and glutamate. The
pathway generates obligatory substrates for the synthesis of
glycoproteins and glycolipids; major HNSP products that accumulate in
cells are UDP-N-acetyl hexosamines
(UDP-HexNAc), i.e., UDP-N-acetyl
glucosamine (UDP-GlcNAc) and
UDP-N-acetyl galactosamine (UDP-GalNAc) in an ~3:1 ratio (reviewed in Refs. 19, 25). Glucosamine
(GlcN) enters cells via the same transporter as glucose and is rapidly
phosphorylated to GlcN-6-P, bypassing
GFAT. GlcN has been extensively used to demonstrate that accumulation
of HNSP products can induce insulin resistance of glucose transport and
glycogen synthesis in skeletal muscle and fat, both in vitro and in
vivo (4, 19, 25, 27).
The major site of glucose-induced insulin resistance is skeletal muscle
(10). Prolonged hyperglycemia results in the accumulation of UDP-HexNAc
in muscle (26), and insulin clamp studies in rats under various
experimental conditions suggest a relationship between the development
of insulin resistance and increased UDP-HexNAc in muscle (13, 14).
Transgenic mice, overexpressing GFAT in muscle and fat, develop insulin
resistance with age (15). In two other models of insulin resistance,
the ob/ob mouse and transgenic mice
overexpressing the glucose transporter isoform GLUT-1 in muscle, GFAT
activity and UDP-HexNAc concentrations in muscle are also increased (6,
7).
The growth hormone (GH)-deficient dwarf (dw) rat arose as an autosomal
recessive mutation in the Lewis (Lw) rat. Although the gene defect has
not been identified, the phenotype has been well characterized (8). GH
synthesis and storage are reduced by ~90% in dw rats, whereas the
production of other pituitary hormones is normal (8). These animals
manifest decreased growth rate and body weight, which is reversible
with GH treatment (8, 28, 32). GH-releasing factor mRNA is increased in
the hypothalamus of dw rats, and somatotroph cell number is markedly
reduced (reviewed in Ref. 5). Somatotrophs isolated from dw rats show
decreased ability to increase cAMP production in response to various
stimuli. The impairment in maximal cAMP production, although not
affecting acute GH release, may underlie the defect in somatotroph cell number and GH content in the dw pituitary (5). Because chronic GH
excess causes insulin resistance (reviewed in Ref. 29), and GH
deficiency may enhance insulin sensitivity (21, 30), we examined
whether HNSP activity was affected in muscles of this GH-deficient
animal model.
Animals.
Homozygous dw rat breeding pairs (dw-4-ola-hsd) were obtained from
Harlan Olac (Bicester, UK) and bred (32). Because of significant sexual
dimorphism of GH production in rats, only male offspring were used for
experiments. Because the original mutation was observed in Lw rats,
age-matched male Lw rats were obtained from Harlan Sprague
Dawley (Indianapolis, IN) and used as controls (32). Rats were housed
in controlled animal facilities (23°C, 12:12-h light-dark cycle);
Lw rats were acclimated to the laboratory for
ABSTRACT
Top
Abstract
Introduction
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
References
INTRODUCTION
Top
Abstract
Introduction
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
References
RESEARCH DESIGN AND METHODS
Top
Abstract
Introduction
RESEARCH DESIGN AND METHODS
RESULTS
DISCUSSION
References
1 wk before use.
Animals were fed standard rat chow ad libitum, unless otherwise
indicated, and had free access to water at all times. In some
experiments food was withdrawn for 18 h before animals were killed
(i.e., fasted). In other experiments, controls were pair fed with dw
rats; i.e., animals were housed in individual cages, and the amount of
food consumed by dw rats per gram of body weight on 1 day was offered
to control rats (adjusted for body weight) on the following day. Food
was withdrawn from all rats 2 h before they were killed.
Analysis of nucleotide-linked hexoses and hexosamines.
The method used and its validation have been described previously (26).
Briefly, frozen tissue powder was homogenized at 4°C in 0.3 M
perchloric acid (PCA), precipitates were pelleted by centrifugation,
and PCA was extracted from the supernatant with 1:4
trioctylamine:1,1,2-trichloro-trifluoro-ethane. The aqueous phase was
stored at 70°C until analysis by high-performance liquid chromatography (HPLC) within 1 wk. HPLC was performed on a Whatman Partisil anion exchange column (4.6 × 250 mm) eluted with a
concave gradient of ammonium phosphate from 15 mM, pH 3.8, to 1 M, pH 4.5, over 50 min at a flow rate of 1 ml/min. UDP-HexNAc, UDP-hexoses (UDP-Hex), GDP-mannose, and UDP were quantified by ultraviolet absorption (A254) and comparison
with standards. With this method, UDP-GlcNAc coelutes with UDP-GalNAc,
and UDP-glucose coelutes with UDP-galactose. The ratio of glucose to
galactose containing sugar nucleotides is typically 3:1, and their
absorption coefficients are very similar (26). Therefore, UDP-GlcNAc
and UDP-glucose were used as standards for UDP-HexNAc and UDP-Hex, respectively.
GFAT enzyme activity.
GFAT activity was assayed as previously described (6, 26). Frozen
tissue powder was homogenized in 4-5 vol of extraction buffer
[25 mM HEPES, pH 7.5, 4°C, 5 mM EDTA, 100 mM KCl, 5 mM glucose 6-phosphate, and protease inhibitors (6)]. Extracts were
centrifuged at 4°C (60,000 g for
15 min, and the supernatant was centrifuged at 100,000 g for 60 min). The supernatant was spin-filtered over Sephadex G-25 columns equilibrated with assay buffer
(25 mM
K2PO4,
pH 7.5, 1 mM EDTA, 50 mM KCl) to reduce the concentration of small
molecules that may modify GFAT activity. Aliquots of gel-filtered
cytosolic extracts were incubated for 60 min at 37°C with 6 mM
fructose 6-phosphate and 12 mM glutamine. Reactions were stopped with
PCA, samples were centrifuged, and PCA was extracted from the
supernatants as we have described. The aqueous phase was stored at
70°C until analysis within 1 wk.
GlcN-6-P, the product of the
GFAT-catalyzed reaction, was measured fluorometrically after separation
by HPLC, as described in Ref. 26. Blank readings were <10% of
measured activity and were subtracted. GFAT activity was normalized to
the protein concentration in the gel-filtered extract, which was
measured spectrophotometrically using Coomassie protein assay reagent
(Pierce, Rockford, IL) and bovine serum albumin standards. GFAT
activity was expressed as picomoles of
GlcN-6-P generated per milligram of
protein per minute.
Measurement of plasma glucose and insulin. Plasma glucose was determined by the glucose oxidase method using a Beckman Glucose Analyser II. Plasma insulin was measured by a radioimmunoassay kit for rat insulin (Linco Research, St. Charles, MO).
Statistical analyses. Means ± SE are shown. Where error bars are not shown, they are too small for graphic representation (i.e., <3% of the mean). The significance of differences between means was analyzed by two-tailed Student's t-test. When multiple means were compared, analysis of variance with post hoc analysis by Tukey's test to accommodate unequal sample sizes was used. Regression analysis with indicator variables was used to assess the relationship between age and GFAT, UDP-HexNAc, and UDP-Hex. P < 0.05 was considered statistically significant.
Materials. Reagents used were of the highest purity available and were purchased from Sigma or as indicated in the text.
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RESULTS |
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Animal characteristics.
Male dw rats and controls were studied between 5 and 14 wk of age. Dw
rats weighed 26% less than controls at 5 wk of age
(P < 0.01), 34% less than controls
between 8 and 10 wk (P < 0.001), and
40% less than controls between 12 and 14 wk
(P < 0.001, Fig. 1). Dw rats consumed less food than the
controls at each age studied, but food intake was not significantly
different between groups when normalized to body weight (~8
g · 100 g body
wt1 · day
1,
Fig. 1). Concentrations of plasma glucose and insulin were
measured in 9- to 10-wk-old control and dw rats in the fed and fasted
states. Plasma insulin concentrations were higher in fed than in fasted rats but were not significantly different between control and dw rats
in either condition. Plasma glucose tended to be slightly lower in dw
rats than in controls, but the difference (11%) was significant only
in the fasted state (n = 9, P < 0.02, Fig. 2).
|
|
Developmental changes.
GFAT activity and nucleotide sugar concentrations in muscle were
measured in male control and dw rats at various ages between 5 and 14 wk (Table 1). GFAT activity in muscle was
greatest in 5-wk-old prepubertal rats and decreased with maturation.
Regression analysis revealed that muscle GFAT activity decreased with
age in dw and in control rats, (r = 0.8285
and
0.673 for dw and control rats, respectively;
P < 0.0001 for both,
n = 28 and 31, respectively). There
was no significant difference in muscle GFAT activity between 5-wk-old
control and dw rats. Muscle GFAT activity was 30-40% lower in dw
rats than in controls at 8-10 wk of age (P < 0.01) and 60% lower than in
controls at 12-14 wk (P < 0.01). Dw rats and age-matched controls were always studied in the same experiment and analyzed in parallel.
|
Comparison of fed and fasted rats. Because GFAT activity in muscle may be regulated by the ambient concentrations of glucose and insulin (9), we examined whether the differences between dw and control rats were dependent on the nutritional state (Fig. 2). Nine to ten-week-old dw and control rats were either fasted overnight or fed ad libitum, with feed withdrawn 2 h before experiments. GFAT activity in muscle was 40% lower in dw rats than in controls, both in the fed and the fasted states (P < 0.01 in both conditions, Fig. 2). Similarly, UDP-HexNAc concentrations in muscle were 30% lower in dw rats than in controls under both fed (P < 0.01) and fasted (P < 0.05) conditions (Fig. 2). The concentrations of UDP-Hex were similar in muscles from dw and control rats in the fed and fasted states (Fig. 2). The ratio of UDP-HexNAc to UDP-Hex was 1.82 ± 0.2 and 1.87 ± 0.1 in fasted and fed controls, respectively, and 1.41 ± 0.1 in both fasted and fed dw rats (P < 0.01 dw vs. control). UDP concentrations in muscle were similar in control and dw rats in the fed and fasted states (data not shown). GDP-mannose concentrations in muscle were significantly lower in dw rats vs. controls, but only in the fasted state (6.2 ± 0.61 vs. 9.1 ± 0.79 nmol/g muscle, P < 0.01).
Liver.
To test whether the decreased HNSP activity in muscles of dw rats was
tissue specific, GFAT activity and the concentrations of nucleotide
sugars were measured in livers from 5-, 8-, and 10-wk-old rats. As
previously reported (26), GFAT activity and the concentrations of
nucleotide sugars were much higher in liver than in muscle (compare
Fig. 3 with Fig. 2). In contrast to muscle, no marked age-associated changes in GFAT activity were observed in
liver. In 5-wk-old rats, hepatic GFAT activity was 810 ± 50 pmol · mg
protein1 · min
1
in controls vs. 780 ± 69 pmol · mg
protein
1 · min
1
in dwarf rats (n = 3/group). When data
from 8- to 10- wk-old rats were pooled for analysis
(n = 7/group, Fig. 3), hepatic GFAT activity was slightly lower in the dw animals (14.5%,
P < 0.05) than in the controls. No
differences in the level of UDP-HexNAc or UDP-Hex were observed between
livers of control and dw rats in any age group.
|
Treatment with rhGH or rhIGF-I.
To assess whether the decreased HNSP activity observed in muscles of dw
rats was reversible with GH treatment, ad libitum-fed, 10- to 14-wk-old
dw rats were injected subcutaneously with 100 µg rhGH twice daily, at
9 AM and 8 PM, for 2 or 5 days. In each experiment, treated and
untreated control and dw rats were of the same age and were analyzed in
parallel. In some experiments, an additional set of untreated control
rats was pair fed to the untreated dw rats on a weight-adjusted basis.
Control rats were treated with GH for only 2 days. To facilitate data
presentation, measurements in the different groups were expressed as a
percentage of the mean value observed in untreated control rats from
the same experiment (Fig. 4). Pair feeding
did not affect GFAT activity or nucleotide sugar concentrations in
muscle (Fig. 4). There were no significant differences in food intake
per gram body weight between any of the groups (data not shown).
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DISCUSSION |
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The inherited defect in homozygous dw rats is a paucity of pituitary somatotrophs, which results in a marked (~90%) reduction in pituitary GH content and secretion. The responsiveness of somatotrophs to GH-releasing hormone (GHRH) and somatostatin and the sex-specific pulsatility of GH secretion are maintained, although the amplitude of individual pulses is markedly reduced in dw rats (5, 17). Circulating IGF-I is 25-30% of that observed in controls (28, 32). The metabolic abnormalities observed in this model are direct or indirect consequences of GH deficiency. Therefore, our data suggest that GH participates in the regulation of HNSP in skeletal muscle in young, postpubertal male rats. This conclusion is supported by the consistent reduction in GFAT activity in muscles of 8- to 14-wk-old dw rats, as well as the reduced concentration of the major products of HNSP, UDP-HexNAc. Furthermore, treatment with rhGH for 5 days restored UDP-HexNAc concentrations in muscles of dw rats to the level observed in controls and appeared to partially restore GFAT activity. Of interest is the apparent tissue specificity of HNSP regulation by GH, i.e., dw rats showed a greater percent reduction in GFAT activity in muscle than in liver, and UDP-HexNAc concentrations were reduced only in muscle. From these data, we conclude that GH deficiency reduces HNSP activity in skeletal muscle more than in liver. Similar tissue specificity was previously observed in ob/ob mice, where GFAT activity and UDP-HexNAc concentrations in muscle were significantly greater than in lean controls, without concomitant hepatic changes (6).
During the first 3 wk of life, there are no significant differences in growth rate between control and dw rats, but growth differences are clearly manifest between 3 and 5 wk of age (Ref. 8 and Fig. 1). In a preliminary study (n = 3/group), we did not detect significant differences in muscle HNSP activity between 5-wk-old control and dw rats. The emergence of differences between 5 and 8 wk of age (Table 1) coincides with the onset of puberty in rats, which is accompanied by a fall in somatostatin mRNA, a rise in GHRH mRNA expression in the hypothalamus, and the appearance of high-amplitude GH secretory pulses in male rats (1, 17).
Developmental regulation of GFAT activity has been observed in many rodent tissues (24). In general, GFAT activity in fetal or neoplastic tissues is higher than that in adults. It is 10-fold lower in muscles of adult rats than in fetal muscle tissue (24). In cultured myocytes derived from human skeletal muscle, GFAT activity is an order of magnitude higher than in human muscle biopsies (9). A gradual decline in muscle GFAT activity and UDP-HexNAc concentrations in muscles between 8 and 14 wk of age has been observed in mice (6). In the present study, the major decline of GFAT activity in rat muscle occurred between 5 and 9 wk, when circulating GH tends to be high (1, 17). Therefore, although our data indicate that GH acts as a positive modulator of GFAT activity and hexosamine synthesis in muscle, other unidentified factors may act as negative regulators during development. To our knowledge there is no information concerning possible sex-related differences in HNSP regulation in muscle.
The age-dependent decline in muscle UDP-HexNAc concentrations was smaller than that of GFAT activity (see Table 1), supporting the concept that glucose flux via HNSP reflects several metabolic parameters in addition to GFAT activity. These include the rate of glucose transport into the cell (7, 19) and the activity of competing pathways of glucose metabolism, such as glycolysis and glycogen synthesis (14, 26). Furthermore, UDP-GlcNAc is a potent inhibitor of GFAT activity (see reviews in Refs. 19, 26), which would dampen product accumulation in vivo. It is also possible that the downstream metabolism of UDP-HexNAc is developmentally regulated.
Our results with GH and IGF-I therapy suggest that GH rather than IGF-I regulates HNSP activity in muscle. In previous studies, which compared the effects of rhGH and rhIGF-I in dw rats, GH was much more effective than IGF-I in stimulating bone growth, although changes in body weight were similar, and serum IGF-I was higher in IGF-I-treated than in GH-treated dw rats (8). However, GH and IGF-I exert differential effects on the expression of IGF-I binding proteins and on IGF-I expression in muscle of dw rats (18). GH infusion enhances IGF-I expression in muscles of dw rats primarily by inducing IGF-I production in fibroblasts residing between muscle fibers (31). GFAT activity in muscle mainly reflects GFAT expression by muscle cells (23). Thus, although IGF-I may participate via autocrine or paracrine mechanisms in the regulation of HNSP by GH in muscle, a direct effect of circulating IGF-I seems unlikely.
GH exerts dual effects on glucose metabolism in muscle. A rapid,
transient, insulin-like effect appears to be mediated by tyrosine
phosphorylation of insulin receptor substrate (IRS) 1 by janus kinase 2 (JAK2), which associates with and is activated by the dimerized,
occupied GH receptor (2, 11). Prolonged GH treatment results in insulin
resistance, as manifested by increased circulating insulin
concentrations, decreased glucose utilization by muscle, increased
lipolysis, and eventually hyperglycemia (reviewed in Ref. 29). The
mechanism of this insulin resistance is poorly understood. Insulin
receptor number is increased in muscle, GLUT-4 expression and
subcellular distribution are unchanged, but GLUT-1 expression is
diminished (22, 29) in the presence of prolonged GH exposure. An
impairment in insulin-stimulated phosphorylation on tyrosine residues
of the insulin receptor -subunit and of IRS-1 has been proposed as a
cause of GH-induced insulin resistance (29), and impaired activation of
muscle glycogen synthase by insulin has been reported (3). In our
studies, plasma glucose concentrations were similar in nonfasted dw
rats with or without GH treatment for 5 days. Mean plasma insulin was
higher in the GH-treated group, but the difference was not significant
(data not shown). UDP-glucose is the obligatory substrate of glycogen synthase, and the concentration of UDP-Hex in muscle reflects the rate
of glucose transport and phosphorylation, on one hand, and the rate of
UDP-glucose utilization, (i.e., glycogen synthesis) on the other
(reviewed in Ref. 26). The fact that UDP-Hex increased significantly in
muscles of dw rats after 5 days of GH treatment suggests that glycogen
synthase activity may have been reduced. Increased fatty acid (FFA)
oxidation promotes glucose flux via HNSP, possibly by inhibiting
glycolytic flux at the level of phosphofructokinase (14). FFA oxidation
in muscle is increased by GH (3, 21) and may thereby contribute to
increased UDP-HexNAc concentrations in muscles of GH-treated dw rats.
Whether GH deficiency increases insulin sensitivity is controversial
(21, 30) and has not been investigated in dw rats. Although in this
study fasting glycemia was significantly lower in dw rats than in
controls, neither the serum insulin concentrations nor the
insulin-to-glucose ratios were altered significantly (Fig. 2 and data
not shown, respectively).
HNSP has been implicated in mediating the induction of certain growth
factors in response to hyperglycemia in vascular smooth muscle cells
and in renal tubular and mesangial cells (reviewed in Refs. 16, 20). Of
particular interest is a recent report indicating that increased flux
via HNSP may mediate the hyperglycemia-induced overexpression of
transforming growth factor-1 in
mesangial cells, which in turn leads to increased matrix production
(16). Dw rats are in great part protected from developing the renal
complications of diabetes, i.e., renal and glomerular hypertrophy and
microalbuminuria (12). Immunocytochemical studies suggest that GFAT
protein may be induced in glomerular epithelial and mesangial cells by
uncontrolled diabetes (23). Whether GH participates in the regulation
of HNSP in the kidney needs to be established.
In summary, the data presented indicate that GH acts as a positive regulator of HNSP in skeletal muscle. Whether GH's effect is direct or is mediated by muscle-derived IGF-I or is secondary to other more direct effects of GH on intermediary metabolism is not known. Assuming that our findings in a GH-deficient model may apply to conditions with chronic GH excess, one may speculate that increased glucose flux via HNSP may contribute to GH-induced insulin resistance.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the gift of rhIGF-I by Genentech to S. M. Willi. We thank Drs. Nancy Wright and Lyndon Key for providing the dwarf rats used in initial experiments and breeding, Dr. Key for the generous gift of rhGH, and Jeffrey S. Koning for excellent technical assistance with some of the analyses. We thank Hillarie Stecker for excellent secretarial assistance and Barbara Wojciechowski for expert statistical advice and analysis.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02001.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. G. Buse, Dept. of Medicine, Division of Endocrinology, Medical Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425-2222.
Received 29 April 1998; accepted in final form 6 November 1998.
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