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Am J Physiol Endocrinol Metab 288: E973-E979, 2005. First published December 21, 2004; doi:10.1152/ajpendo.00549.2004
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Adipocytes with increased hexosamine flux exhibit insulin resistance, increased glucose uptake, and increased synthesis and storage of lipid

Donald A. McClain,1,2 Mark Hazel,2 Glendon Parker,2 and Robert C. Cooksey1,2

1Veterans Affairs Medical Center and 2Department of Medicine, University of Utah, Salt Lake City, Utah

Submitted 15 November 2004 ; accepted in final form 17 December 2004


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The hexosamine signaling pathway has been shown to serve a nutrient-sensing function. We have previously shown that overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine-fructose-6-phosphate amidotransferase) in adipose tissue of transgenic mice results in skeletal muscle insulin resistance and altered regulation of leptin and adiponectin. To dissect the pathways by which the hexosamine pathway affects fuel storage and energy homeostasis, we have examined the characteristics of adipocytes from these animals. After 3 mo of age, epididymal fat pads from adult transgenic animals are 42% heavier (P = 0.003) and individual adipocytes are 23% larger in diameter (P < 0.05) than those from littermate wild-type controls. Isolated adipocytes from transgenic mice are insulin resistant, with a 2.5-fold increase in the ED50 for stimulation of 2-deoxy-D-glucose uptake. However, maximal insulin-stimulated glucose uptake is increased in transgenic adipocytes by 39% (P < 0.05). This upregulation of glucose uptake was associated with a 41% increase in the expression of GLUT4 mRNA and a 28% increase in GLUT4 protein in transgenics compared with controls (P < 0.05). GLUT1 mRNA and protein did not significantly differ between fasted control and transgenics. Total lipid synthesis was also increased in epididymal adipocytes from transgenic animals by 206% compared with controls (P < 0.05). Fatty acid oxidation was increased 1.6-fold in the transgenic adipocytes (P < 0.05). We conclude that the hexosamine signaling pathway upregulates fat storage in adipocytes in states of carbohydrate excess, in part by increasing GLUT4 and glucose uptake and by augmenting fatty acid synthesis.

hexosamine; insulin resistance; adipocyte

ALTHOUGH THERE IS A MAJOR GENETIC CONTRIBUTION to type 2 diabetes, the largest predisposing factor remains caloric excess and/or obesity. Underlining the importance of this mechanism, excess glucose and lipids not only result from but also cause the pathological hallmarks of diabetes, insulin resistance and {beta}-cell failure. One pathway by which excess nutrients can contribute to the diabetic phenotype is the hexosamine biosynthesis pathway. This pathway has been shown to mediate nutrient sensing in several tissues and cell culture models, and chronic hexosamine excess mimics many of the key aspects of type 2 diabetes syndrome, including insulin resistance, hyperinsulinemia, hyperlipidemia, hyperleptinemia, and obesity (4, 11, 19, 31, 37, 40, 42). Thus it has been hypothesized that the hexosamine pathway acts as a nutrient sensor that participates in directing excess calories to storage as fat.

One of the hallmarks of type 2 diabetes is insulin resistance. We have generated transgenic mice that overexpress the rate-limiting enzyme for hexosamine biosynthesis, glutamine-fructose-6-phosphate amidotransferase (GFA), in muscle and adipose tissue under control of the GLUT4 promoter (11) or in adipose tissue alone under control of the adipocyte-specific fatty acid-binding protein (aP2) promoter (10). Both mouse models exhibit insulin resistance and decreased uptake of glucose into skeletal muscle, although they do not develop diabetes. The work that first implicated the hexosamine pathway in insulin resistance described downregulation of glucose uptake after glucosamine treatment of isolated adipocytes (19). This result was consistent with numerous descriptions of adipocyte glucose uptake in human type 2 diabetes and its animal models that also demonstrate downregulation of glucose uptake and the insulin-stimulatable glucose transporter GLUT4 (2, 7, 8). However, if the hexosamine pathway normally plays a physiological role in stimulating diversion of calories for storage as fat in situations of ingestion of excess calories, then augmented glucose uptake in adipocytes might be predicted at the earlier stages of nutrient excess. When we first examined isolated adipocytes from the GLUT4-GFA transgenic mice, we had noted that basal and maximal glucose uptake rates were actually somewhat increased compared with controls (unpublished data). However, because these animals overexpressed GFA in both fat and muscle, we could not rule out an effect of cross talk between those tissues as opposed to an intrinsic effect of hexosamine excess in the adipocyte. We therefore have examined glucose uptake in adipocytes isolated from mice overexpressing GFA only in adipocytes. We find that epididymal fat pads and individual adipocytes are larger in the transgenic animals despite normal serum glucose, triglyceride, and nonesterified fatty acid levels. Glucose uptake and GLUT4 are upregulated in the adipocytes of these animals, although there is decreased insulin sensitivity for stimulation of glucose uptake. Fat synthesis is also augmented. The results are consistent with a nutrient-signaling role for the hexosamine pathway in adipocytes. Furthermore, they suggest that downregulation of GLUT4 in diabetic fat cells may be a later result of the diabetic state and not related to excess carbohydrate per se.


   EXPERIMENTAL PROCEDURES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Routine reagents were purchased from Sigma (St. Louis, MO), unless otherwise indicated.

Transgenic animals. GFA transgene expression was targeted to adipose tissue using a 5.4-kb aP2 promoter generously provided by Dr. Bruce Spiegelman (9) linked to the human GFA cDNA (45) and the SV40 polyadenylation sequence. These mice have been previously characterized as being insulin resistant, hyperleptinemic, and hypoadiponectinemic in the fasting state (10). The transgenic descendants from a single male founder have been bred onto a C57BL6 background for greater than seven generations. Mice were fed a diet based on soy protein and with 10% kcal as fat. They were kept on a 12:12-h dark-light cycle, and 12-h fasting was initiated 4 h after initiation of the dark (feeding) cycle. Heterozygous transgenic mice and control wild-type animals from the same litters were used in experiments that were approved by the Laboratory Animal Use Committees at the University of Utah Medical Center and the Salt Lake City Veterans Affairs Medical Center. Data from both male and female animals were pooled for analysis, unless otherwise noted.

Quantitation of mRNA by RT-PCR. Epididymal fat pads from mice, either fasted 24 or 6 h after being refed, were dissected, placed in 800 µl RNA-Later (Ambion, Austin, TX), and stored at –20°C. The fat pad (70 mg) was placed in 1.4 ml Tri Reagent (MRC, Cincinnati, OH), shredded in a homogenizer for 10 s, and homogenized using a Sonic Dismembrator-60 (setting 6 for 5 s; Fisher Scientific, Springfield, NJ). RNA was then prepared according to the manufacturer's protocol and dissolved in 40 µl FORMazol (MRC). RNA concentrations were measured spectrophotometrically. First-strand cDNA synthesis was carried out using 1.3-µg samples of RNA and 300 ng of random hexamer primers (Invitrogen, Carlsbad, CA) in a reaction volume of 25 µl, using Superscript II RT (Invitrogen) according to the manufacturer's protocol, with the exception that the final dithiothreitol concentration was 2 mM (15).

Real-time PCR was performed with a rapid thermal cycler (LightCycler; Roche Diagnostics) using a modification of a published protocol (25). Reactions were performed using 8 ng cDNA as template. Final concentrations of PCR reagents were as follows: 0.5 µM each primer, 200 µM each dNTP, 50 mM Tris, pH 8.3, 500 µg/ml nonacteylated BSA (Sigma), 3.0 mM MgCl2, 0.04 U/µl Platinum Taq DNA polymerase (Invitrogen), and 1:30,000 dilution of SYBR Green I fluorescent dye (Molecular Probes, Eugene, OR). Primers based on murine sequences were chosen using the Primer3 program (32). For GLUT4, 5'-CCTGAGAGCCCCAGATACCTCTAC (sense) and 5'-GTCGTCCAGCTCGTTCTACTAAG (antisense) amplified a 380-bp product. For GLUT1, 5'-gctgggaatcgtcgttgg and 5'-GATGGGCTGGCGGTAGG amplified a 334-bp product. For cyclophilin-A, 5'-AGCACTGGAGAGAAAGGATTTGG and 5'-tcttcttgctggtcttgccatt amplified a 349-bp product. Amplification used 26–45 four-step cycles, with the rate of temperature change between steps of 20°C/s. Steps were 95°C with a 0-s hold, 60°C with a 0-s hold, 72°C with a 11-s hold, and 80°C with a 1-s hold. Fluorescence was detected during the fourth step (at a temperature previously determined to be slightly below the melting temperature of the PCR products). After amplification, a melting curve was generated by slowly heating the double-stranded DNA product. Analyses of the postamplification melting curves and visualization of the DNA products after agarose gel electrophoresis confirmed the absence of nonspecific DNA products. For each fluorescence amplification curve, the second derivative maximum was determined using the LightCycler software. Standardization and normalization were based on a published method (34). Standard curves of log cDNA vs. second derivative maximum (fractional cycle number) were constructed for each transcript from cDNA mixes comprised of equal amounts of cDNA from each subject. Results for each individual cDNA were normalized by dividing its relative amount by the amount of cyclophilin-A determined in the same LightCycler run for the same subject and using the same PCR "cocktail" but with the cyclophilin primers.

2-Deoxy-D-glucose uptake into isolated adipocytes in vitro. Mice were killed and weighed, and the epididymal fat pads were excised and weighed. For each experiment, two to three transgenic and littermate control animal fat pads were pooled in 12 ml Hank's balanced salt solution (HBSS)-4% BSA with 20 mg collagenase (Calbiochem). The fat pads were minced with scissors, and the suspension was shaken in a gyratory water bath at 180 rpm for 50 min at 37°C. The cells were strained through 100-µm nylon mesh in 50-ml tubes, the netting was rinsed with two times with ~10 ml HBSS-1% BSA, and the cells were centrifuged at 880 rpm for 3 min at 20°C. After the infranate was aspirated, the cells were gently resuspended in 8 ml HBSS-1% BSA and recentrifuged as above. After the infranate was aspirated, the cells were gently resuspended in 2.8 ml HBSS-1% BSA. The cell suspension (640 µl) was added to 60 µl of insulin to yield final concentrations of 0, 0.5, 1, 10, or 100 nM. The cell suspension (100 µl) was also added to 100 µl of 2% OsO4 for fixation and cell counting. Cells were incubated with insulin for 30 min at 37°C in a gyratory bath rotating at 180 rpm. 2-deoxy-D-[3H]glucose (final 1 mM 2-deoxy-D-glucose, 2 µCi/tube) was added in 50 µl, and after 5 min cells were harvested by adding 250 µl of the cell suspension to 100 µl of di-isononyl phthalate in 400-µl microfuge tubes. The cells were centrifuged for 10 s at 14,000 rpm in a microfuge. The cells (top layer) were cut away from the buffer (bottom layer) and put in scintillation tubes, and radioactivity was determined by scintillation counting. Background radioactivity was determined by counting cells exposed to radioactivity and immediately harvested. Each cell incubation was assayed in duplicate. An aliquot of the adipocytes was fixed by osmium tetroxide as described (5), and cell size was determined by light microscopy in a hemocytometer with a calibrated eyepiece.

Assay of fatty acid oxidation and total lipid synthesis. The oxidation of palmitate was assayed as described (24). Adipocytes were prepared as above, and 1-ml aliquots of cells were cultured in HBSS-1% BSA for 2 h with 2 µCi [3H]palmitic acid (53 Ci/mmol; Amersham Biosciences, Piscataway, NJ). Release of tritium in water was measured by adding 1 ml of 100% TCA and then 5 ml of chloroform to the cell suspension. After centrifugation (3,000 g, 3 min), 1 ml of the aqueous fraction was added to 1 ml of 5% dextran-charcoal and recentrifuged as above. Tritium release in an aliquot was measured by liquid scintillation counting. Background tritium release, as measured in an aliquot of medium with palmitate that was incubated without cells, was subtracted from the experimental values. For determination of total lipid synthesis, 1-ml aliquots of cells were cultured in HBSS-1% BSA with 250 µCi [3H]H20. After 5 h, 5 ml of water-saturated chloroform were added to the cultures. The mixture was vortexed for 1 min and centrifuged, and 4 ml of the chloroform were transferred to a fresh tube and extracted with 5 ml water. The chloroform phase (2 ml) was dried, and tritium incorporation was measured by liquid scintillation counting. Background tritium extraction in the chloroform phase was measured in an aliquot of medium that had been incubated without cells and was subtracted from the experimental values.


   RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General characteristics of the transgenic animals. Mice overexpressing GFA in fat under control of the aP2 promoter have been previously described (10). Fasted transgenic mice were found to have mild glucose intolerance with normal serum glucose, insulin, and triglycerides but skeletal muscle insulin resistance as analyzed in vivo by the euglycemic-hyperinsulinemic clamp technique. The fasted transgenic animals exhibited decreased serum adiponectin and increased serum leptin levels compared with wild types. We therefore sought to analyze the properties of the adipocytes of these animals to better understand the role of hexosamines in regulating fat metabolism.

The transgenic mice did not weigh significantly more than their control littermates, although after 3 mo of age a trend toward increased weight did emerge (Fig. 1A, P = 0.09). In younger male mice, there was a trend toward larger epididymal fat pads that was not statistically significant (Fig. 1B, P = 0.10). After 3 mo of age, however, the epididymal fat pads of male transgenic mice were 42% heavier (Fig. 1B, P = 0.003) and accounted for a larger fraction of total body weight (data not shown, P = 0.02). The increased fat mass could be mainly accounted for by increased fat cell size rather than adipocyte hyperplasia. The adipocytes from the transgenic animals were 24% larger in diameter than wild-type adipocytes (Fig. 1C, P < 0.01), and there was no significant difference in the numbers of cells recovered after collagenase digestion of the fat pads (Fig. 1D). The increase in fat tissue of the transgenic mice was also seen as increased interstitial (not intramyocellular) fat noted in histological analysis of skeletal muscle, noted in double-blinded analysis by a histopathologist not involved in this study (data not shown). The weight of the intrascapular brown fat pads did not differ between transgenic and control animals (data not shown), and subcutaneous fat depots were not visibly increased.



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  		Fig. 1. Body weights and characteristics of epididymal fat depots. A: body weights were determined in mice aged 2–3 and 4–6 mo of age (n = 20–32/group, all males). B: epididymal fat pads from mice were excised and weighed before use in the experiments shown in C and D. After collagenase digestion, aliquots of the cells were fixed in osmium tetroxide. Cell size (C) and number (D) were determined by counting cells in a hemocytometer and measuring them using a calibrated ocular. *P < 0.05 by t-test.

 
Isolated adipocytes from transgenic animals have increased levels of basal and insulin-stimulated glucose uptake but decreased insulin sensitivity. It has been previously reported that in vitro exposure of adipocytes to glucosamine results in insulin resistance, namely decreased insulin sensitivity for stimulation of glucose uptake and decreased responsiveness to insulin (19). We therefore examined the insulin-stimulated uptake of 2-deoxy-D-glucose in isolated adipocytes in vitro. Adipocytes from transgenic animals were insulin resistant, as indicated by the rightward shift in the insulin dose-response curve (Fig. 2A). Analysis of the concentration of insulin resulting in half-maximal stimulation in each experiment revealed a 2.5-fold increase in that value for transgenic cells compared with cells from littermate control animals (0.7 ± 0.1 nM in controls, 1.8 ± 0.2 in transgenic, n = 4, P < 0.05). However, basal and maximally insulin-stimulated levels of glucose uptake were increased in transgenic animals by 22 and 39%, respectively (Fig. 2B, P < 0.005). Similar results (a trend toward increased uptake but insulin resistance manifest by a rightward shift in the dose-response curve) were obtained using adipocytes from animals expressing GFA under control of the GLUT4 promoter (11), i.e., overexpressing GFA in adipocytes as well as skeletal and cardiac muscle (data not shown).



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  		Fig. 2. Insulin resistance for glucose uptake in isolated adipocytes. Glucose uptake was measured in epididymal adipocytes isolated by collagenase digestion as described. A: dose-response curves for insulin-stimulated glucose uptake. Cells were exposed to the indicated concentrations of insulin. Maximal uptake (100%) was determined at 100 nM insulin. Results are the means ± SE of 4 independent experiments. The concentration of insulin resulting in half-maximal uptake was determined by interpolation from each individual experiment. That concentration was significantly increased in the aP2 transgenic mice (1.8 ± 0.2 nM) compared with wild types (0.7 ± 0.1 nM, P < 0.05). B: total 2-deoxy-D-glucose (2-DOG) uptake was measured in the absence (basal) or presence (maximum) of 100 nM insulin. Results are means ± SE of 5 separate experiments, each using pooled adipocytes from 2–3 mice 3–6 mo of age, and each assayed in duplicate. *P < 0.005 by t-test.

 
To explore the possible explanations for increased glucose uptake, we determined the levels of mRNA and protein for the glucose transporters GLUT1 and GLUT4. GLUT1 mRNA assessed by quantitative RT-PCR (Fig. 3A) and GLUT1 protein assessed by Western blotting (Fig. 3B) were not altered in the transgenic mice. GLUT4 mRNA was increased by 46% in fasted transgenic mice compared with fasted wild types (Fig. 3A, P < 0.05), and that was paralleled by a 28% increase in GLUT4 protein (Fig. 3B, P < 0.05).



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  		Fig. 3. Levels of mRNA and protein for the glucose transporters GLUT1 and GLUT4. A: mRNA was purified from epididymal fat pads of control and aP2-glutamine-fructose-6-phosphate amidotransferase mice that had fasted for 12 h. cDNA was made from each fat pad sample using random hexanucleotide priming. PCR was performed on 50 ng of each cDNA sample using primers specific for the indicated cDNAs and cyclophilin A. Results for each cDNA were normalized to the levels of cyclophilin A, and the level of each mRNA in control mice was set at unity. Results are means ± SE of determinations from 4 (transgenic) or 6 (control) separate fat pads, each from a different mouse. B: GLUT1 and -4 protein levels were determined by Western blotting using extracts from 5 wild-type and aP2 mice. *P < 0.05 by t-test.

 
Fatty acid oxidation and lipid synthesis are both increased in adipocytes that overexpress GFA. Oxidation of palmitic acid and total fat synthesis, measured by the incorporation of tritium from water in chloroform-extractable material, were next determined in isolated adipocytes. As shown in Fig. 4, fat synthesis was increased 2-fold and fatty acid oxidation 1.6-fold in adipocytes from aP2-GFA transgenic mice compared with wild-type littermates (P < 0.05 for both). We examined the mRNAs for several key enzymes involved in fat synthesis and oxidation (fatty acid synthase and the medium-, long-, and very long-chain fatty acyl-CoA dehydrogenases) and found no significant changes between the transgenic and wild-type mice that accounted for the observed differences (data not shown).



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  		Fig. 4. Increased total fat synthesis and fatty acid oxidation in adipocytes from aP2 transgenic mice. Results for fat synthesis are expressed as dpm tritium incorporated in the chloroform-extractable phase (x10–3). Results for fatty acid oxidation are dpm from tritiated palmitate released in water, also x10–3. Results are the means of 4 independent comparisons, each from an individual mouse (3–6 mo of age), with each experiment assayed in duplicate. *P < 0.05 by t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously reported that mice overexpressing GFA, the rate-limiting enzyme for hexosamine synthesis, in fat plus muscle are insulin resistant for glucose uptake in skeletal muscle in vivo (4, 11). Fat-specific overexpression of GFA is sufficient to cause this phenotype (10). These results confirm the role of the hexosamine pathway in nutrient sensing and insulin resistance and, more specifically, in the redistribution of excess calories from muscle to fat in situations of excess feeding (20, 31). In the current study, we have examined in greater detail the response of the adipocyte to excess nutrient flux as signaled by increases in hexosamine flux. This response is to increase GLUT4 expression, glucose uptake, and fat synthesis, resulting in increased adiposity and adipocyte size. One caveat to the conclusions drawn is that the results derive from a single founder line of transgenic mice. Thus it is possible that some of the effects are the result of nonspecific effects related to the site of transgene insertion. However, as stated in a previous publication describing these mice (10), the reported results are most likely specific for GFA overexpression because the phenotype is consistent with that observed when GFA is overexpressed in muscle plus fat under control of the GLUT4 promoter, and the phenotype is also not attributable to interference with endogenous aP2 transcription.

Marshall et al. (19) had first implicated the hexosamine pathway in insulin resistance based on the requirement for glutamine and the ability of glucosamine to downregulate glucose transport in primary cultures of rat adipocytes. In that model, however, there was a decrease in insulin responsiveness (maximal glucose uptake) and decreased sensitivity to insulin. Similarly, either downregulation (38) or no change (26) of GLUT4 has been observed in 3T3-L1 cells exposed acutely to glucosamine plus insulin. The reason for these discordances, namely the lack of decreased glucose uptake or downregulation of GLUT4 with chronic overexpression of GFA, is not clear. The most obvious differences between the studies are the time span and degree to which the cells experience increased hexosamine flux. The effects of hexosamines are mediated by O-linked glycosylation of cytosolic and nuclear proteins and thus exert their effects by both posttranslational and transcriptional mechanisms (3, 22, 28, 43). For example, the Munc18 protein involved in GLUT4 vesicle trafficking is posttranslationally affected by O-linked glycosylation and has been hypothesized to play a role in the hexosamine-induced inhibition of GLUT4-mediated glucose uptake. Such effects might be predominant in short-term glucosamine treatment, whereas transcriptional effects such as the upregulation of GLUT4 might be more evident with chronic upregulation of the pathway. Furthermore, the aP2-GFA transgenic mice exhibit increased hexosamine flux within the range that occurs in diabetes or with dietary manipulation, such as high-fat feeding (6), and thus are likely to represent physiological changes. In contrast, acute treatment of cells in vitro with glucosamine can lead to much higher levels of hexosamine products. In extreme cases, glucosamine treatment can lead to depletion of cellular ATP (12), although the latter is not seen in most in vivo or transgenic models (4). Thus it would be expected that the balance of multiple posttranslational and transcriptional events would be different in the transgenic animals compared with animals exposed to acute glucosamine treatments, possibly accounting for the differences in the phenotypes observed.

The regulation of GLUT4 seen in animal models of overfeeding, genetic obesity, and diabetes is complex, and the effects of these manipulations on GLUT4 expression or activity are tissue and diet specific (13). This is not necessarily surprising, given the different needs of muscle vs. fat as energy consumer vs. storage depot, or the different strategies an organism would employ in high-fat vs. high-carbohydrate overfeeding. The mechanisms employed to regulate GLUT4 also vary across tissues. GLUT4 activity in diabetic muscle, for example, is more affected by posttranslational mechanisms than in fat, where transcriptional mechanisms dominate (14). Furthermore, levels of glycemia, fatty acids, or insulin also play a role in regulation. For example, hypoinsulinemic models of diabetes would be predicted to (and do) have different effects on GLUT4 regulation than hyperinsulinemic models or models engendered by overfeeding. Finally, adipocyte GLUT4 levels in many of these animal models are not static. In the Zucker fa/fa rat, for example, GLUT4 upregulation is seen early in obesity, whereas when frank diabetes ensues GLUT4 is downregulated (30). With these complexities in mind, it is possible nevertheless to integrate the current findings into the larger body of work on GLUT4 regulation in the adipocyte. The results are very consistent with the Zucker fa/fa model early in the overfeeding stage (~5 wk of age) when there are increased GLUT4 protein and mRNA (on a per cell basis) and increased levels of basal and insulin-stimulated glucose uptake (30). As diabetes ensues later in the fa/fa model, glucose uptake decreases like it does in human diabetes, late obesity, and high-fat feeding (7, 8, 29). Thus the current model of adipocyte-specific GFA overexpression best mimics carbohydrate overfeeding and early obesity before the confounding variables of frank diabetes, relative hypoinsulinemia, or hyperlipidemia have ensued. Consistent with this conclusion, the animals from which these adipocytes were derived had normal serum levels of insulin, triglycerides, nonesterified fatty acids, and glucose.

The contribution to obesity of increased glucose flux in adipocytes is consistent with the observation that transgenic GLUT4 overexpression also leads to obesity (36, 39). These investigators observed marked increases in lipogenesis and de novo fatty acid synthesis that were 20- to 30-fold increased over nontransgenic controls (36). Conversely, when GLUT4 is decreased in GLUT4+/– heterozygous knockout mice, fat pad weight tends to be lower than in controls (16). Not all of the effects of GFA overexpression, however, are likely to be mediated by GLUT4. For example, the changes in lipogenesis (2-fold) we observed with modest (41%) GLUT4 upregulation are proportional to those observed in the studies cited above, wherein much more robust changes in lipogenesis (20- to 30-fold) were seen in the face of much greater GLUT4 overexpression (6- to 9-fold; see Refs. 36 and 39). However, the changes in fat accretion were not proportional in the two models, and GLUT4 overexpression by itself leads to adipocyte hyperplasia rather than hypertrophy (36). Thus other mechanisms are likely to be operative in the aP2-GFA mice, including effects mediated by paracrine action of dysregulated adipokines (10). Of note, in a previous publication describing these mice (10) we did observe insulin resistance at 2–3 mo of age, before the weights of the fat pads of the transgenic mice had diverged from controls, suggesting that the effects of hexosamine flux on insulin resistance are independent of adiposity or fat cell size. Thus the current results are consistent with the hypothesized role for hexosamines as integrated nutrient sensors (20, 31). That is, the pathway is hypothesized to be used to measure intracellular "satiety" and would be predicted to lead to an organismwide shift toward pathways that favor energy storage in fat. This is observed when GFA is overexpressed in muscle plus fat, in the liver, or in {beta}-cells (11, 37, 40) or when the hexosamine pathway is activated by glucosamine infusion in the intact rat (27). Thus, in circumstances of physiological excess in nutrient availability, it would not necessarily be predicted nor adaptive for fat to markedly downregulate glucose uptake.

We also observed a marked increase in total fat synthesis in isolated adipocytes from the aP2-GFA mice, and this is consistent with the increase in adipocyte size and adiposity in the model. The mechanism for the increased fat accretion is likely to be multifactorial, with only part of the increase explained by the modest increase in glucose uptake. The expression of mRNAs for both fatty acid synthase and acetyl-CoA carboxylase, for example, has been reported to be upregulated by acute increases in hexosamine flux (33). Although we did not see a consistent upregulation of either mRNA in our more chronic model, there may have been multiple hormonal influences and/or other compensatory changes over time to blunt any apparent changes in these mRNAs. Somewhat paradoxically, we also observed an increase in fatty acid oxidation in the transgenic adipocytes with increased hexosamine flux. Thus there may be some degree of futile cycling of fatty acids analogous to the futile cycling of glucose that is elevated in diabetes (23). Futile cycling of fatty acid intermediates is observed in yeast grown in 2% glucose (18). Although the mechanism is not completely understood, regulated release of short- or medium-chain fatty acyl-CoAs from fatty acid synthase may be involved. We have also observed an activation of AMP-dependent kinase by hexosamines, a strategy that would allow fatty acid oxidation to proceed even in the face of nutrient (and energy) sufficiency (unpublished observation).

Current data suggest that hexosamines regulate metabolism through the mechanism of O-linked glycosylation of nuclear and cytosolic proteins by the addition of serine- or threonine-linked N-acetylglucosamine (3). The levels of the end product of the hexosamine pathway, UDP-N-acetylglucosamine, are limiting for this modification of proteins by cytosolic O-glycosyltranferase (1, 17, 35, 44). Direct evidence for the role of O-glycosylation in metabolic regulation has recently emerged. Pharmacological inhibition of the enzyme responsible for removal of O-linked N-acetylglu cosamine, leading to increased levels of O-glycoylation, results in insulin resistance in cultured adipocytes (41). Transgenic overexpression of O-glycosyltransferase under control of the GLUT4 promoter also leads to insulin resistance and hyperleptinemia, mimicking the phenotype seen when GFA is overexpressed in the same tissues (21). We have previously shown that the levels of O-linked N-acetylglucosamine protein modification are increased in the aP2-GFA transgenic fat pads (10), although the identity and the functional significance of the modified proteins are not yet established. O-linked N-acetylglucosamine modification regulates protein activity both directly (posttranslationally) and through transcriptional mechanisms, and the relative contribution of these mechanisms to the phenotype of the aP2-GFA transgenic adipocytes is not known.

The current data add to a growing body of evidence that hexosamines are used by tissues to sense the nutrient status of the organism to coordinate changes in cell growth and metabolism. The pattern of changes seen with overexpression of GFA in several tissues (hyperinsulinemia, muscle insulin resistance, increased glycogen and fat synthesis in the liver) can be seen as an adaptive response of cells to direct excess calories to storage as fat (4, 11, 37, 40). However, these same pathways can also have detrimental consequences, especially when chronically stimulated, including insulin resistance, obesity, hyperlipidemia, {beta}-cell failure, and type 2 diabetes. A longitudinal study of the mice with overexpression of GFA targeted to fat indicates that these mice do develop glucose intolerance and gain excess weight as they age (10). Use of these and other models of increased hexosamine flux should aid in understanding the mechanisms underlying these detrimental consequences of chronic overnutrition and their link to type 2 diabetes. The current studies also point to a direct role of the adipocyte in these processes.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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 GRANTS
REFERENCES
 
This work was supported by the Research Service of the Verterans Administration, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43526, and the Ben and Iris Margolis Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. McClain, Dept. of Medicine, 30 N. 2030 East, Univ. of Utah School of Medicine, 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
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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