AJP - Endo Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 290: E334-E340, 2006. First published September 27, 2005; doi:10.1152/ajpendo.00265.2005
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/E334    most recent
00265.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cooksey, R. C.
Right arrow Articles by McClain, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cooksey, R. C.
Right arrow Articles by McClain, D. A.

Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in {beta}-cells

Robert C. Cooksey, Sumitha Pusuluri, Mark Hazel, and Donald A. McClain

Veterans Administration Medical Center and Division of Endocrinology, Metabolism and Diabetes, Department of Internal Medicine, University of Utah, Salt Lake City, Utah

Submitted 13 June 2005 ; accepted in final form 23 September 2005


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hexosamines serve a nutrient-sensing function through enzymatic O-glycosylation of proteins. We previously characterized transgenic (Tg) mice with overexpression of the rate-limiting enzyme in hexosamine production, glutamine:fructose-6-phosphate amidotransferase, in {beta}-cells. Animals were hyperinsulinemic, resulting in peripheral insulin resistance. Glucose tolerance deteriorated with age, and males developed diabetes. We therefore examined islet function in these mice by perifusion in vitro. Young (2-mo-old) Tg animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM and half maximal at 9.9 ± 0.5 mM glucose in Tg islets compared with maximal at 30 mM and half maximal at 13.5 ± 0.7 mM glucose in wild type (WT; P < 0.005). Young Tg animals secreted more insulin in response to 20 mM glucose (Tg, 1,254 ± 311; WT, 425 ± 231 pg·islet–1·35 min–1; P < 0.01). Islets from older (8-mo-old) Tg mice became desensitized to glucose, with half-maximal secretion at 16.1 ± 0.8 mM glucose, compared with 11.8 ± 0.7 mM in WT (P < 0.05). Older Tg mice secreted less insulin in response to 20 mM glucose (Tg, 2,256 ± 342; WT, 3,493 ± 367 pg·islet–1·35 min–1; P < 0.05). Secretion in response to carbachol was similar in WT and Tg at both ages. Glucose oxidation was blunted in older Tg islets. At 5 mM glucose, islet CO2 production was comparable between Tg and WT. However, WT mice increased islet CO2 production 2.7 ± 0.4-fold in 20 mM glucose, compared with only 1.4 ± 0.1-fold in Tg (P < 0.02). Results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, acting at least in part by modulating glucose oxidation pathways. Prolonged excess hexosamine flux results in glucose desensitization and mimics glucose toxicity.

islet; desensitization; glucose sensing

GLUCOSE METABOLISM through the hexosamine biosynthesis pathway serves as a nutrient-sensing function and has been shown to mediate many of the adverse effects of hyperglycemia, or so-called "glucose toxicity." This hypothesis has been supported by studies in diverse experimental systems, including cultured cells and intact animals treated with either glucosamine or genetically engineered alterations in the hexosamine signaling pathway (3, 5–7, 10–12, 21, 29, 32, 37). The rate-limiting enzyme in the hexosamine biosynthesis pathway is glutamine:fructose-6-phosphate amidotransferase (GFA), which catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. The terminal metabolites of the hexosamine pathway are uridyl diphosphate (UDP)-hexosamines. UDP-N-acetylglucosamine (UDP-GlcNAc) is a substrate for the cytosolic UDP-GlcNAc peptide glycosyltransferase [or O-glycosyltransferase (OGT)], which glycosylates nuclear and cytosolic proteins with a single N-acetylglucosamine moiety on serine and threonine residues (14, 20). Recently, this O-linked glycosylation (O-GlcNAc) has been shown to mediate the effects of stimulating the hexosamine pathway (23, 28, 38).

A pattern of results in these experimental model systems is that hexosamines signal a shift in gene expression and enzymatic activities that favor long-term energy storage in the organism. Over time, many of the model animals also develop characteristics of the type 2 diabetes syndrome. We have previously reported (35) that overexpression of GFA targeted to {beta}-cells results in peripheral hyperinsulinemia. In response, the animals become modestly insulin resistant, and with age the male mice develop diabetes. To further explain these findings, we have performed studies on isolated islets from the mice expressing GFA under control of the rat insulin promoter (RIP). We demonstrate that hexosamine flux initially sensitizes the cells to glucose-induced insulin secretion, but as the animals age, the cells become desensitized. This is associated with a defect in glucose oxidation in the islets.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Routine reagents were purchased from Sigma (St. Louis, MO) unless otherwise indicated.

Transgenic animals. Transgenic mice on a C57BL6 background with GFA targeted to pancreatic {beta}-cells with the RIP have been described previously (35). Mice that were heterozygous for the transgene were compared with nontransgenic wild-type mice as controls. Mice were genotyped by polymerase chain reaction using transgene-specific primers. All procedures were approved by the Institutional Animal Care and Use Committee.

Isolation of islets. Islets of Langerhans were isolated from fasted wild-type and transgenic mice by collagenase digestion (Roche Applied Science, Indianapolis, IN), as described in the kit instructions. The islets were then individually handpicked from acinar tissue.

Islet perifusion. Islets were perifused as previously described (45, 46). Briefly, groups of size-matched islets (10–15/group) were isolated and placed on a 62-µm monofilament nylon mesh (Small Parts, Miami Lakes, FL) and inserted in 13-mm filter holders (Swinnex, Millipore, Bedford, MA). Islet groups were perifused (1 ml/min) in Krebs-Ringer bicarbonate buffer (NaCl, 126 mM; KCl, 4.8 mM; KH2PO4, 1.2 mM; MgSO4-7H2O, 1.2 mM; CaCl2, 2.2 mM; NaHCO3, 5.0 mM; HEPES, 10 mM; glucose, 2.8 mM; BSA, 1%, pH 7.4). The buffer was gassed with 95% O2-5% CO2 and maintained at 37°C. After a 30-min equilibration period, the islet groups were perifused with 3, 5, 10, 20, or 30 mM glucose or 10 mM glucose plus 10 µM carbachol and 1-ml fractions of the perfusate collected for an additional 40 min. Insulin concentration in the perfusate was determined by radioimmunoassay (Sensitive Rat Insulin RIA Kit; Linco Research, St. Charles, MO).

Quantitative RT-PCR analysis of mRNA. Between 20 and 72 isolated islets per mouse from six transgenic and five wild-type (sibling), C57BL/6-strain background, 8-mo-old male mice were collected, submerged in 250 µl of RNA-Later (Ambion, Austin, TX), and stored at –20°C. Each islet sample was diluted with 200 µl of neutral PBS and centrifuged (2,500 rcf for 1 min at 4°C). The supernatant was gently removed, and 250 µl of Tri Reagent (MRC, Cincinnati, OH) were added. RNA isolation steps were performed according to the manufacturer's protocol, except that the tissue was homogenized using a Sonic Dismembrator 60 (Fischer Scientific, setting 2.5 for 24 x 0.5-s pulses). Each RNA pellet was suspended in 40 µl of FORMazol (stabilized formamide, MRC). RNA yields were between 1.1 and 2.8 µg/sample. First-strand cDNA synthesis used Superscript II reverse transcriptase (Invitrogen Life Technologies), according to that manufacturer's protocol, with 380 ng of islet RNA as described (16).

Quantitative PCR was performed with a Roche LightCycler using Primer3-designed primers as previously described (4), except that the cycle's annealing step was set at 60°C for all primers. Six nanograms of cDNA (based on an assumed 1:5 mass ratio of RNA input to cDNA output during cDNA synthesis with Superscript II RT) was used per 10 µl of PCR reaction. Additional primers (forward and then reverse) for the following amplicons used were succinate dehydrogenase A 331 bp: 5'-CTGTGAAAGGCTCCGACTGG, 5'-GCAATGACACCACGGCACT; succinate dehydrogenase B 327 bp: 5'-GCAAAGTCTCCAAAATCTACCCTCTT, 5'-GGCGTTCCTCTGTGAAGTCGT; aconitase 319 bp: 5'-GTGGGTGGTGATTGGAGATGA, 5'-CTGGGTCTCGTTGAAGGTGTG; citrate synthase 345 bp: 5'-TTTCAGACCCTTACCTGTCCTTTG, 5'-GGTTCTTAGCCTTCCCTTGCTC; malate dehydrogenase (MDH) 323 bp: 5'-AGACTGTGCCCTTCCCCTTC, 5'-GAGATTTTGCTCGGTTGTGGTC; NADH-ubiquinone oxidoreductase MWFE subunit (Ndu1a) 303 bp: 5'-GAGTAACGGTGCGGAGATGTG, 5'-GCTGCATAGCCTTCTAACAGGAAC; NADH-ubiquinone oxidoreductase B22 subunit (Ndu1b) 312 bp: 5'-TGCTGAGGGAGGCAGAAGAA, 5'-ACAGTGGGGGCAAGTCACC; ATP synthase subunit-{epsilon} (ATP5{epsilon}) 312 bp: 5'-GGGCCGGTTTGAGGCTAC, 5'-CCCAGGAGGTGAGGTTGATTT; mitochondria-encoded ATP synthase subunit (mit-ATP6) 324 bp: 5'-CAAACAAATAATGCTAATCCACACACC, 5'-GCTGTAAGCCGGACTGCTAATG; RPL13a 373 bp: 5'-CCCGAGGCCCCTACCAC, 5'-CTTGCAGATTTTCTTCTCCACATTCT.

Standard curves of log cDNA vs. crossing point cycle number were constructed for each transcript in parallel as part of the same LightCycler run, using a titration (of 2, 4, 6, 8, and 10 ng of cDNA) of a compilation of all 11 cDNA samples (33). Results for the 12 transcripts of interest listed in Table 1 represent the relative per-mouse values, which were averaged from duplicate or quadruplicate PCR reactions and divided by the averaged per-mouse normalizer transcript values (cyclophilin 349 bp and RPL13a 373 bp). Then, per-genotype normalized values were averaged and expressed as the transgenic-to-wild type ratio.


View this table:
[in this window]
[in a new window]
  		Table 1. Islet mRNA levels in WT and RIP-GFA Tg mice

 
Glucose oxidation. Groups of 15–40 size-matched islets per condition were collected and placed in microfuge tubes containing 1 ml of Krebs-Ringer buffer, which had been gassed (95% O2-5% CO2) and supplemented with glucose (5 mM), BSA (0.1%), and HEPES (10 mM, pH 7.5). The islets were centrifuged (100 rcf for 2 min), and the supernatant was decanted and replaced with 100 µl of oxygenated buffer in either 5 or 20 mM glucose plus 3 µCi of D-[U-14C]glucose (302 mCi/mmol, Amersham). The islets were placed in 20-ml glass scintillation vials, oxygenated (95% O2-5%CO2) for 5 min, capped, and shaken at 37°C for 120 min. Islet metabolism was stopped with an injection of 100 µl of antimycin A (0.05 mM) into the medium, followed by an injection of 750 µl of hyamine hydroxide (Packard Instruments) into the scintillation vial. 14CO2 was liberated from the medium with a subsequent injection of 100 µl of Na2HPO4 (0.4 mM, pH 6.0) into the medium and absorbed by the hyamine hydroxide. The islet tube was removed from the vial, scintillation fluid was added, and radioactivity was measured.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characteristics of transgenic animals. The phenotype of mice overexpressing GFA under control of the RIP has been described previously (35). To summarize those published results, GFA activity was increased by 76% in transgenic islets. Islet ATP content, body weights, and serum triglycerides did not differ between wild-type and transgenic mice. Serum levels of fatty acids were slightly lower in the transgenic mice. Young (2-mo-old) transgenic animals exhibited hyperinsulinemia (64–65% higher serum insulin levels in the fasting and postchallenge states) with normal fasting glucose and slightly lower glucose excursions after intraperitoneal glucose challenge than their wild-type littermates. Older (8–10 mo) transgenic females exhibited a slight trend toward higher glucose excursions, whereas in the transgenic males the glucose levels during an intraperitoneal glucose tolerance test were significantly higher than in their wild-type littermates. Weights also increased by 10–15% in the transgenic mice as they aged. In the face of the deterioration of glucose tolerance, insulin levels did not increase in the older animals.

Insulin secretion in isolated islets. The results summarized above suggest altered regulation of insulin secretion from {beta}-cells exposed to excess hexosamine flux. To examine this further, we isolated islets from normal and transgenic mice and studied insulin secretion at various glucose concentrations. Islets from young (2-mo-old) mice exhibited enhanced insulin secretion at 20 mM glucose (Fig. 1A). Significantly more insulin was secreted in both the first and second phases, with the incremental area under the curve threefold higher in islets from the RIP-GFA transgenic mice (Fig. 1B). Both the first and second phases of insulin secretion were similarly increased. Insulin secretion was examined at various concentrations of glucose and is presented as the fold increase over basal secretion as a function of glucose concentration (Fig. 1C). The sensitivity for glucose-induced insulin secretion was greater in the transgenic islets. In wild-type islets, maximal insulin secretion was reached at 30 mM glucose, and half-maximal secretion at 13.5 ± 0.7 mM glucose. In RIP-GFA islets with increased hexosamine flux, secretion was maximal at 20 mM and half-maximal at 9.9 ± 0.5 mM glucose (Fig. 1D, P < 0.005). Total insulin content per islet was not different in transgenic islets compared with wild type (Fig. 2). These results differ from those originally published by our group (35) in that the islet insulin content was originally reported to be higher in the older transgenic mice. We do not know fully the reason for this descepancy, but we have noted a loss over time of copy number of the concatamerized transgene (not shown).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 1. Perifusion of islets from 2-mo-old mice. A: 4–5 islets from wild-type or rat insulin promoter glutamine:fructose-6-phosphate amidotransferase (RIP-GFA) transgenic mice were exposed to 3 mM glucose in perifusion chambers. Islets from male and female mice were used, with no differences detected as a function of sex. At 35 min, glucose concentration was increased to 20 mM. Fractions were collected every minute for 10 min and then every 5 min thereafter (n = 6/group). B: quantification of data from A. For each perifusion, incremental area under insulin curve above baseline (average value for 3 mM glucose used as baseline) was calculated (n = 6/group; *P < 0.05). C: glucose dose response of insulin secretion. Perifusions were repeated at 10, 20, and 30 mM glucose (n = 5 each). Results are plotted as fold increase over basal secretion (3 mM glucose) for each glucose concentration. D: glucose concentration leading to ED50 of insulin secretion. Data in C were plotted as %maximal secretion, and a regression line was calculated. Interpolated value of glucose at 50% maximal secretion was then determined (*P < 0.005).

 


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2. Insulin content of islets from wild-type and RIP-GFA mice. Islets were collected as for perifusion study. Groups of 4–5 islets were sonicated, and insulin concentration measured (n = 8–10 independent determinations/group).

 
In contrast with islets from 2-mo-old mice, islets from older (8-mo-old) mice exhibited decreased insulin secretion and glucose desensitization. Insulin secretion was 35% lower at 20 mM glucose in islets from the RIP-GFA transgenic islets (P < 0.05; Fig. 3, A and B). Sensitivity to glucose of insulin secretion was also lower in the older transgenic islets. Half-maximal secretion occurs at 16.1 mM glucose in RIP-GFA islets compared with 11.8 mM in wild types (P < 0.05; Fig. 3, C and D). At very high concentrations of glucose, insulin secretion decreased in both wild-type and transgenic islets, but the transgenic islets, in addition to being less sensitive to glucose-induced insulin secretion, were also desensitized to this adverse effect of high glucose. In wild-type mice, secretion fell to 64% of normal at 40 mM glucose but only 82% of normal in transgenic islets (P < 0.05). Insulin content of the islets in the older mice tended to be lower than in wild types, but not significantly so (Fig. 2). Of note, all of the perifusion studies employed islets from both male and female mice, and no differences were noted in insulin secretion as a function of sex. Thus the abnormalities seen in the islets are likely not a function of a secondary response to hyperglycemia, which develops signifcantly only in male mice (35).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 3. Perifusion of islets from 8-mo-old mice. Studies shown in Fig. 1 for 2-mo-old mice were repeated using islets from 8-mo-old mice. Islets from male and female mice were used, with no differences detected as a function of sex. A: 4–5 islets from control or RIP-GFA transgenic mice were exposed to 3 mM glucose in perifusion chambers. At 35 min, glucose concentration was increased to 20 mM. Fractions were collected every minute for 10 min and then every 5 min thereafter; n = 6/group. B: quantification of data from A. For each perifusion, incremental area under insulin curve above baseline (average value for 3 mM glucose used as baseline) was calculated (n = 6/group; *P < 0.05). C: glucose dose response of insulin secretion. Perifusions were repeated at 10, 30, and 40 mM glucose (n = 4–6 each). Results are plotted as fold increase over basal (3 mM glucose) secretion for each glucose concentration. D: glucose concentration leading to ED50 of insulin secretion. Data in C were plotted as %maximal secretion, and a regression line was calculated. Interpolated value of glucose at 50% maximal secretion was then determined (*P < 0.05).

 
Pharmacological stimulation of insulin secretion. To determine whether there was a general defect in insulin secretion, islets were exposed to carbachol. There were no significant differences between transgenic and wild-type islets in levels of carbachol-induced insulin secretion (Fig. 4).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Carbachol-stimulated insulin secretion in control and RIP-GFA islets. Perifusion studies were carried out as described in legends to Figs. 1 and 2, but at 35 min, 10 mM glucose and 10 µM carbachol were added to perifusion buffer. A: 2-mo-old mice (n = 4/group). B: 8-mo-old mice (n = 4/group).

 
Glucose oxidation in isolated islets. The reduced and desensitized insulin secretion observed in the islets of older transgenic mice in response to glucose, but not carbachol, suggests a defect in a more proximal step in glucose sensing. Islets from older mice exhibited a significant decrease in glucose oxidation (Fig. 5). At 5 mM glucose, CO2 production in 8-mo-old islets was comparable between transgenic and wild-type mice (wild type 0.76 ± 0.14, transgenic 0.89 ± 0.08 pmol CO2·islet–1·2 h–1). However, islets from wild-type mice increased CO2 production 2.7 ± 0.4-fold in 20 mM glucose compared with 5 mM glucose, whereas in transgenic islets the increase was only 1.4 ± 0.1-fold (P < 0.02).



View larger version (7K):
[in this window]
[in a new window]
  		Fig. 5. Glucose oxidation in isolated islets from 8-mo-old control and RIP-GFA mice. A: release of 14C label from glucose into CO2 was determined on batches of 15–40 islets, with 8 independent determinations for each glucose concentration in each group of mice. In each study, islets from a single islet preparation were split, with one-half exposed to 5 mM glucose and one-half to 20 mM glucose. B: within each study shown in A, ratio of CO2 production at 20 vs. 5 mM glucose was determined. (*P < 0.02).

 
Assays of select mitochondrial enzyme activities and mRNAs. We next investigated whether the observed decrease in glucose oxidation might be related to changes in mRNAs for proteins either known to be involved in glucose oxidation and deregulated in diabetes or for proteins previously demonstrated to be regulated by hexosamine flux. Several mRNAs for nuclear and mitochondrially encoded enzymes were unaltered in the transgenic islets (Table 1). The list includes several proteins previously shown to be downregulated in the fat cells of glucosamine-treated rats (MDH, Ndu1a, Ndu1b, ATP5{epsilon}) (26). Enzyme activities of citrate synthase, succinate dehydrogenase, and pyruvate carboxylase were also unaltered (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The hallmarks of {beta}-cell dysfunction in type 2 diabetes include early hypersecretion, thought to be a response to insulin resistance, followed by progressive loss of glucose-induced insulin secretion and ultimate loss of {beta}-cell mass. Many of these features are also seen in {beta}-cells exposed to high concentrations of glucose (31). The molecular mechanisms for this so-called "glucose toxicity" are unknown, but we have shown previously (35) that some of these effects can be reproduced in islets that overexpress GFA, which is the rate-limiting enzyme in hexosamine synthesis. In the present work, we have extended these observations and reported that excess hexosamine flux results initially in augmentation of glucose-induced insulin secretion, but with aging there is progressive desensitization of insulin secretion accompanied by a defect in glucose oxidation. The hexosamine pathway of glucose metabolism also contributes to insulin resistance (3, 5–7, 10, 12, 21, 29, 32), obesity (11, 37), and hyperlipidemia (37) and has been hypothesized to function as a cellular satiety sensor responsible for damping acute insulin signaling and facilitating the storage of excess nutrients as fat (22, 30, 41). The pathway exerts its effects through the substrate-limited O-GlcNAc of nuclear and cytosolic proteins by the enzyme OGT (23, 28, 38).

There have been several studies that have addressed the effects of hexosamine flux on {beta}-cells. Shankar et al., for example, showed that glucosamine infusion into intact rats led to a blunting of both glucose- and arginine-stimulated insulin secretion (34). It was later shown that glucosamine treatment or GFA overexpression in isolated rat islets also resulted in decreased insulin secretion and downregulation of insulin gene expression (13). In the latter case, however, an inhibitor of enzymatic deglycosylation did not mimic GFA overexpression, leading the authors to conclude that O-GlcNAc did not mediate the effects. Rather, it was found that glucosamine could increase hydrogen peroxide levels, leading the authors to conclude that oxidant stress was responsible. Another report (50) demonstrated that inhibition of the hexosamine pathway led to decreased insulin secretion from mouse islets, suggesting that the pathway might trigger biphasic responses (stimulatory and inhibitory) on insulin secretion depending on the degree or chronicity of stimulation. Other reports (1, 8, 18) have demonstrated {beta}-cell death and mitochondrial dysfunction in glucosamine-treated {beta}-cells. Some caution should be used in interpreting the studies using glucosamine exposure, however, in that glucosamine in high concentrations can inhibit glucokinase and glucose transporter 2 (GLUT2)-mediated glucose transport (2, 36). Overexpression of GFA, by contrast, would not be expected to generate significant amounts of free glucosamine and has not done so in animal models (12). One possible complication of the present study is the known expression of insulin promoter-targeted genes in the hypothalamus, although this should have relatively little impact on the phenotype of the isolated islets.

The mechanism for the decrement of glucose-induced insulin secretion in older islets that overexpress GFA likely involves decreased glucose oxidation. Beyond that result, however, the detailed molecular mechanism remains unclear. It has been reported that hexosamine flux can increase the DNA-binding activity of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) (9) that includes as its targets the genes for glucokinase, GLUT2, and insulin (17, 24, 27, 39, 40). This represents one mechanism by which the increase in glucose-stimulated insulin secretion might occur in the younger animals.

The older transgenic animals exhibit a loss of glucose-induced insulin secretion, and an obvious question arises as to whether both contradictory responses could be mediated by the same hexosamine pathway. The same genes whose expression would be stimulated by glycosylation of PDX-1 have also been observed to be downregulated in islets with high hexosamine flux (13, 44). Consistent with this, we also observed a trend toward downregulation of GLUT2 and the insulin2 gene in the older transgenic islets. The modest nature of these changes might be related to the relatively low degree of GFA overexpression in the islets (35). In animal models of GFA overexpression in several tissues, we have experienced a consistent difficulty in achieving high levels of overexpression, which is perhaps related to the proapoptotic effects of the pathway (8, 18, 19, 25, 42). This might also explain the lack of significant decreases in the levels of message for several other mRNAs reported to be downregulated by the hexosamine pathway (26), although those effects might also be restricted to the adipocytes where the phenomenon was observed. Other defects, such as impaired phosphoinositide signaling, have also been observed in {beta}-cells treated with glucosamine (47). Thus it is likely that a panoply of effects mediated by transcriptional regulation and posttranslational modification of proteins by O-GlcNAc may contribute to the observed decrease in {beta}-cell function induced by high hexosamine flux. This would not be surprising, given the hundreds of proteins that are known to be modified by O-GlcNAc (43). The breadth and complexity of the response to O-GlcNAc might also explain the diverse results that were observed as a function of whether the increased hexosamine flux is acute or chronic or of large or small magnitude. As one example of the competing and disparate effects of hexosamine signaling, overexpression of the pathway has been reported to both induce oxidant stress (13) and protect from oxidant stress (49). Such apparently contradictory effects could be rationalized teleologically; if the hexosamine pathway is a sensor for high nutrient fluxes, then a rational response to those fluxes would be to upregulate the capacity for mitochondrial oxidation while at the same time upregulating antioxidant defense mechanisms. The net result in any tissue would then reflect the oxidant burden vs. the degree of upregulation of antioxidant defenses. These would presumably vary across tissues, with nutrient burden and, perhaps as in the present study, with the age of the host. At this time, however, we cannot rule out an alternative hypothesis, namely that the primary effect of the hexosamine pathway is glucose sensitization, whereas the desensitization is a secondary response, for example, to the hyperglycemia or insulin resistance that develops in the model. The latter hypothesis is made less likely by the finding that the islets from both male and female mice exhibit the glucose-insensitive phenotype, even though the female mice exhibit only very mild glucose intolerance (35).

In summary, we have demonstrated that hexosamine flux in {beta}-cells exerts a biphasic effect, initially stimulating but then blunting glucose-induced insulin secretion through the inhibition of glucose oxidation. These effects parallel features of type 2 diabetes and suggest that excess nutrient flux may exert some of their adverse effects through this pathway. This hypothesis is supported by the observed association with diabetes of polymorphisms in genes encoding key enzymes in the hexosamine pathway (15, 48).


   GRANTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Research Service of the Veterans Administration, the National Institute of Diabetes and Digestive and Kidney Diseases (DK-43235), and the Ben B. and Iris M. Margolis Foundation.


   ACKNOWLEDGMENTS
 
We acknowledge Walter Zawalich of Yale University for generous assistance with the perifusion studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. McClain, Division of Endocrinology and Metabolism, University of Utah School of Medicine, 30 N. 2030 East, Salt Lake City, UT 84132 (e-mail: donald.mcclain{at}hsc.utah.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anello M, Spampinato D, Piro S, Purrello F, and Rabuazzo AM. Glucosamine-induced alterations of mitochondrial function in pancreatic {beta}-cells: possible role of protein glycosylation. Am J Physiol Endocrinol Metab 287: E602–E608, 2004.[Abstract/Free Full Text]
  2. Balkan B and Dunning BE. Glucosamine inhibits glucokinase in vitro and produces a glucose-specific impairment of in vivo insulin secretion in rats. Diabetes 43: 1173–1179, 1994.[Abstract]
  3. Baron AD, Zhu JS, Zhu JH, Weldon H, Maianu L, and Garvey WT. Glucosamine induces insulin resistance in vivo by affecting GLUT 4 translocation in skeletal muscle. Implications for glucose toxicity. J Clin Invest 96: 2792–2801, 1995.[Web of Science][Medline]
  4. Cooksey RC, Jouihan HA, Ajioka RS, Hazel MW, Jones DL, Kushner JP, and McClain DA. Oxidative stress, beta-cell apoptosis, and decreased insulin secretory capacity in mouse models of hemochromatosis. Endocrinology 145: 5305–5312, 2004.[CrossRef][Web of Science][Medline]
  5. Crook ED, Daniels MC, Smith TM, and McClain DA. Regulation of insulin-stimulated glycogen synthase activity by overexpression of glutamine: fructose-6-phosphate amidotransferase in rat-1 fibroblasts. Diabetes 42: 1289–1296, 1993.[Abstract]
  6. Crook ED and McClain DA. Regulation of glycogen synthase and protein phosphatase-1 by hexosamines. Diabetes 45: 322–327, 1996.[Abstract]
  7. Crook ED, Zhou J, Daniels M, Neidigh JL, and McClain DA. Regulation of glycogen synthase by glucose, glucosamine, and glutamine:fructose-6-phosphate amidotransferase. Diabetes 44: 314–320, 1995.[Abstract]
  8. D'Alessandris C, Andreozzi F, Federici M, Cardellini M, Brunetti A, Ranalli M, Del Guerra S, Lauro D, Del Prato S, Marchetti P, Lauro R, and Sesti G. Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells. FASEB J 18: 959–961, 2004.[Abstract/Free Full Text]
  9. Gao Y, Miyazaki J, and Hart GW. The transcription factor PDX-1 is post-translationally modified by O-linked N-acetylglucosamine and this modification is correlated with its DNA binding activity and insulin secretion in min6 beta-cells. Arch Biochem Biophys 415: 155–163, 2003.[CrossRef][Web of Science][Medline]
  10. Giaccari A, Morviducci L, Zorretta D, Sbraccia P, Leonetti F, Caiola S, Buongiorno A, Bonadonna RC, and Tamburrano G. In vivo effects of glucosamine on insulin secretion and insulin sensitivity in the rat: possible relevance to the maladaptive responses to chronic hyperglycaemia. Diabetologia 38: 518–524, 1995.[Web of Science][Medline]
  11. Hazel M, Cooksey RC, Jones D, Parker G, Neidigh JL, Witherbee B, Gulve EA, and McClain DA. Activation of the hexosamine signaling pathway in adipose tissue results in decreased serum adiponectin and skeletal muscle insulin resistance. Endocrinology 145: 2118–2128, 2004.[Abstract/Free Full Text]
  12. Hebert LF Jr, Daniels MC, Zhou J, Crook ED, Turner RL, Simmons ST, Neidigh JL, Zhu JS, Baron AD, and McClain DA. Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance. J Clin Invest 98: 930–936, 1996.[Web of Science][Medline]
  13. Kaneto H, Xu G, Song KH, Suzuma K, Bonner-Weir S, Sharma A, and Weir GC. Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress. J Biol Chem 276: 31099–31104, 2001.[Abstract/Free Full Text]
  14. Kreppel LK, Blomberg MA, and Hart GW. Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats. J Biol Chem 272: 9308–9315, 1997.[Abstract/Free Full Text]
  15. Lehman DM, Fu DJ, Freeman AB, Hunt KJ, Leach RJ, Johnson-Pais T, Hamlington J, Dyer TD, Arya R, Abboud H, Goring HH, Duggirala R, Blangero J, Konrad RJ, and Stern MP. A single nucleotide polymorphism in MGEA5 encoding O-GlcNAc-selective N-acetyl-beta-D glucosaminidase is associated with type 2 diabetes in Mexican Americans. Diabetes 54: 1214–1221, 2005.[Abstract/Free Full Text]
  16. Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM, and Moorman AF. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 307: 63–69, 2002.[CrossRef][Web of Science][Medline]
  17. Leonard J, Peers B, Johnson T, Ferreri K, Lee S, and Montminy MR. Characterization of somatostatin transactivating factor-1, a novel homeobox factor that stimulates somatostatin expression in pancreatic islet cells. Mol Endocrinol 7: 1275–1283, 1993.[Abstract/Free Full Text]
  18. Liu K, Paterson AJ, Chin E, and Kudlow JE. Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death. Proc Natl Acad Sci USA 97: 2820–2825, 2000.[Abstract/Free Full Text]
  19. Liu K, Paterson AJ, Zhang F, McAndrew J, Fukuchi K, Wyss JM, Peng L, Hu Y, and Kudlow JE. Accumulation of protein O-GlcNAc modification inhibits proteasomes in the brain and coincides with neuronal apoptosis in brain areas with high O-GlcNAc metabolism. J Neurochem 89: 1044–1055, 2004.[CrossRef][Web of Science][Medline]
  20. Lubas WA and Hanover JA. Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity. J Biol Chem 275: 10983–10988, 2000.[Abstract/Free Full Text]
  21. Marshall S, Bacote V, and Traxinger RR. Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance. J Biol Chem 266: 4706–4712, 1991.[Abstract/Free Full Text]
  22. McClain DA and Crook ED. Hexosamines and insulin resistance. Diabetes 45: 1003–1009, 1996.[Abstract]
  23. McClain DA, Lubas WA, Cooksey RC, Hazel M, Parker GJ, Love DC, and Hanover JA. Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia. Proc Natl Acad Sci USA 99: 10695–10699, 2002.[Abstract/Free Full Text]
  24. Miller CP, McGehee RE Jr, and Habener JF. IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene. EMBO J 13: 1145–1156, 1994.[Web of Science][Medline]
  25. Nakamura M, Barber AJ, Antonetti DA, LaNoue KF, Robinson KA, Buse MG, and Gardner TW. Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons. J Biol Chem 276: 43748–43755, 2001.[Abstract/Free Full Text]
  26. Obici S, Wang J, Chowdury R, Feng Z, Siddhanta U, Morgan K, and Rossetti L. Identification of a biochemical link between energy intake and energy expenditure. J Clin Invest 109: 1599–1605, 2002.[CrossRef][Web of Science][Medline]
  27. Ohlsson H, Karlsson K, and Edlund T. IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J 12: 4251–4259, 1993.[Web of Science][Medline]
  28. Parker GJ, Lund KC, Taylor RP, and McClain DA. Insulin resistance of glycogen synthase mediated by o-linked N-acetylglucosamine. J Biol Chem 278: 10022–10027, 2003.[Abstract/Free Full Text]
  29. Robinson KA, Sens DA, and Buse MG. Pre-exposure to glucosamine induces insulin resistance of glucose transport and glycogen synthesis in isolated rat skeletal muscles. Study of mechanisms in muscle and in rat-1 fibroblasts overexpressing the human insulin receptor. Diabetes 42: 1333–1346, 1993.[Abstract]
  30. Rossetti L. Perspective: hexosamines and nutrient sensing. Endocrinology 141: 1922–1925, 2000.[Free Full Text]
  31. Rossetti L, Giaccari A, and DeFronzo RA. Glucose toxicity. Diabetes Care 13: 610–630, 1990.[Abstract]
  32. Rossetti L, Hawkins M, Chen W, Gindi J, and Barzilai N. In vivo glucosamine infusion induces insulin resistance in normoglycemic but not in hyperglycemic conscious rats. J Clin Invest 96: 132–140, 1995.[Web of Science][Medline]
  33. Savage DB, Sewter CP, Klenk ES, Segal DG, Vidal-Puig A, Considine RV, and O'Rahilly S. Resistin/Fizz3 expression in relation to obesity and peroxisome proliferator-activated receptor-gamma action in humans. Diabetes 50: 2199–2202, 2001.[Abstract/Free Full Text]
  34. Shankar RR, Zhu JS, and Baron AD. Glucosamine infusion in rats mimics the beta-cell dysfunction of non-insulin-dependent diabetes mellitus. Metabolism 47: 573–577, 1998.[CrossRef][Web of Science][Medline]
  35. Tang J, Neidigh JL, Cooksey RC, and McClain DA. Transgenic mice with increased hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance. Diabetes 49: 1492–1499, 2000.[Abstract]
  36. Uldry M, Ibberson M, Hosokawa M, and Thorens B. GLUT2 is a high affinity glucosamine transporter. FEBS Lett 524: 199–203, 2002.[CrossRef][Web of Science][Medline]
  37. Veerababu G, Tang J, Hoffman RT, Daniels MC, Hebert LF Jr, Crook ED, Cooksey RC, and McClain DA. Overexpression of glutamine:fructose-6-phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage, hyperlipidemia, obesity, and impaired glucose tolerance. Diabetes 49: 2070–2078, 2000.[Abstract/Free Full Text]
  38. Vosseller K, Wells L, Lane MD, and Hart GW. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes. Proc Natl Acad Sci USA 99: 5313–5318, 2002.[Abstract/Free Full Text]
  39. Waeber G, Thompson N, Nicod P, and Bonny C. Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol Endocrinol 10: 1327–1334, 1996.[Abstract/Free Full Text]
  40. Watada H, Kajimoto Y, Miyagawa J, Hanafusa T, Hamaguchi K, Matsuoka T, Yamamoto K, Matsuzawa Y, Kawamori R, and Yamasaki Y. PDX-1 induces insulin and glucokinase gene expressions in alphaTC1 clone 6 cells in the presence of betacellulin. Diabetes 45: 1826–1831, 1996.[Abstract]
  41. Wells L and Hart GW. O-GlcNAc turns twenty: functional implications for post-translational modification of nuclear and cytosolic proteins with a sugar. FEBS Lett 546: 154–158, 2003.[CrossRef][Web of Science][Medline]
  42. Wells L, Whelan SA, and Hart GW. O-GlcNAc: a regulatory post-translational modification. Biochem Biophys Res Commun 302: 435–441, 2003.[CrossRef][Web of Science][Medline]
  43. Whelan SA and Hart GW. Proteomic approaches to analyze the dynamic relationships between nucleocytoplasmic protein glycosylation and phosphorylation. Circ Res 93: 1047–1058, 2003.[Abstract/Free Full Text]
  44. Yoshikawa H, Tajiri Y, Sako Y, Hashimato T, Umeda F, and Nawata H. Glucosamine-induced beta-cell dysfunction: a possible involvement of glucokinase or glucose-transporter type 2. Pancreas 24: 228–234, 2002.[CrossRef][Web of Science][Medline]
  45. Zawalich WS, Bonnet-Eymard M, and Zawalich KC. Glucose-induced desensitization of the pancreatic {beta}-cell is species dependent. Am J Physiol Endocrinol Metab 275: E917–E924, 1998.[Abstract/Free Full Text]
  46. Zawalich WS, Diaz VA, and Zawalich KC. Role of phosphoinositide metabolism in induction of memory in isolated perifused rat islets. Am J Physiol Endocrinol Metab 254: E609–E616, 1988.[Abstract/Free Full Text]
  47. Zawalich WS and Zawalich KC. Glucosamine-induced desensitization of beta-cell responses: possible involvement of impaired information flow in the phosphoinositide cycle. Endocrinology 130: 3135–3142, 1992.[Abstract/Free Full Text]
  48. Zhang H, Jia Y, Cooper JJ, Hale T, Zhang Z, and Elbein SC. Common variants in glutamine:fructose-6-phosphate amidotransferase 2 (GFPT2) gene are associated with type 2 diabetes, diabetic nephropathy, and increased GFPT2 mRNA levels. J Clin Endocrinol Metab 89: 748–755, 2004.[Abstract/Free Full Text]
  49. Zitzler J, Link D, Schafer R, Liebetrau W, Kazinski M, Bonin-Debs A, Behl C, Buckel P, and Brinkmann U. High-throughput functional genomics identifies genes that ameliorate toxicity due to oxidative stress in neuronal HT-22 cells: GFPT2 protects cells against peroxide. Mol Cell Proteomics 3: 834–840, 2004.[Abstract/Free Full Text]
  50. Zraika S, Dunlop M, Proietto J, and Andrikopoulos S. The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets. Arch Biochem Biophys 405: 275–279, 2002.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
Y. A. Soesanto, B. Luo, D. Jones, R. Taylor, J. S. Gabrielsen, G. Parker, and D. A. McClain
Regulation of Akt signaling by O-GlcNAc in euglycemia
Am J Physiol Endocrinol Metab, October 1, 2008; 295(4): E974 - E980.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
E. Ortega, J. Koska, N. Pannacciulli, J. C Bunt, and J. Krakoff
Free triiodothyronine plasma concentrations are positively associated with insulin secretion in euthyroid individuals
Eur. J. Endocrinol., February 1, 2008; 158(2): 217 - 221.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/E334    most recent
00265.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cooksey, R. C.
Right arrow Articles by McClain, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cooksey, R. C.
Right arrow Articles by McClain, D. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.