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CELLULAR METABOLISM
1Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama; 2Department of Pharmacology, College of Osteopathic Medicine, University of New England, Biddeford, Maine; and 3Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 7 August 2006 ; accepted in final form 26 November 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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hexosamine biosynthesis; protein O-glycosylation; O-linked N-acetylglucosamine transferase
There is increasing recognition that the O-GlcNAc modification of serine and threonine residues on cytosolic and nuclear proteins is an important regulatory mechanism involved in signal transduction (20, 34). Unlike other glycosylation events, this reaction occurs in the cytosol and the nucleus rather than in the Golgi or the endoplasmic reticulum and is regulated by the activities of two key enzymes, O-GlcNAc transferase (OGT) and N-acetylglucosaminidase (O-GlcNAcase) (20, 34). The activity of OGT is sensitive to the intracellular concentration of UDP-GlcNAc (20, 34), which is formed by the metabolism of glucose via the hexosamine biosynthesis pathway (HBP). We recently showed (24) that a brief period of streptozotocin (STZ)-induced diabetes blunted the inotropic response of the isolated, perfused heart to phenylephrine, which was reversed by inhibition of the HBP. We also found (24) that acute stimulation of the HBP with glucosamine in normal hearts also blunted the response to phenylephrine and that there was a strong negative correlation between the level of UDP-GlcNAc in the heart and the inotropic response. Previously, Ren et al. (27, 28) showed that incubation of isolated adult cardiomyocytes under hyperglycemic conditions resulted in defects in excitation-contraction (E-C) coupling that were very similar to those seen after STZ-induced diabetes and that this could also be mimicked by incubation with glucosamine. Clark et al. (7) also demonstrated in neonatal cardiomyocytes that increasing protein O-GlcNAc levels either by hyperglycemia or glucosamine treatment resulted in impaired sarcoplasmic reticulum (SR) function. Importantly, they demonstrated that the hyperglycemic effect could be blocked by increasing the expression of O-GlcNAcase and that increased expression of OGT under normal glucose conditions had effects similar to those of hyperglycemia treatment (7). Subsequently, Hu et al. (14) showed that increased O-GlcNAcase expression improved whole heart function in diabetic mice.
Together these studies support the notion that excessive levels of O-GlcNAc may contribute to cardiac dysfunction in diabetes. However, the majority of studies examining the impact of diabetes on E-C coupling have focused on uncontrolled insulin-deficient STZ-induced diabetes, rather than models of Type 2 diabetes. Despite the fact that >90% of patients with diabetes have Type 2 diabetes, relatively little is known about the effect of Type 2 diabetes on cardiomyocyte E-C coupling, and there is no information about the impact of Type 2 diabetes on either O-GlcNAc levels or the expression of OGT, which plays a critical role in regulating O-GlcNAc levels. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, is associated with defects in E-C coupling in adult cardiomyocytes and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression.
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METHODS |
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Animals. Animal experiments were approved by the Institutional Animal Care and Use Committees of the University of Alabama and the University of New England and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, 1996). As a model of Type 2 diabetes, nonfasted male ZDF rats (Gmi-fa/fa) were used with age-matched lean littermates as controls. Animals were maintained on Purina 5008 diet as recommended by the supplier and killed at 6 wk of age or between 21 and 22 wk of age (described as the 22-wk group for clarity). Additional experiments were performed on nonfasted male Sprague-Dawley rats (Charles River Laboratories). In one study, untreated animals were killed at 6 and 22 wk of age. In another study acute insulin-deficient diabetes was induced with intravenous STZ injection (50 mg/kg) in 8-wk-old male Sprague-Dawley rats as previously described (24); vehicle-injected age-matched animals were used as controls. Animals were killed 5 days after treatment.
Analysis of serum metabolites. Blood samples were collected immediately after decapitation from all animals. Serum glucose, insulin, and leptin levels were measured as previously described (35).
Ventricular myocyte isolation, myocyte mechanics, and intracellular Ca2+ transients. Ventricular myocytes were isolated by collagenase digestion as previously described (10). Myocytes were allowed to attach on laminin (10 µg/ml)-coated glass coverslips, maintained in a Tyrode buffer (see below) at 37°C in an incubator, and used within 6 h of isolation. Mechanical properties and intracellular Ca2+ transients of ventricular myocytes were assessed simultaneously with a video-based detection system designed to measure dynamic changes in sarcomere lengths coupled to a fluorescent system (IonOptix, Milton, MA) as described previously (38). Briefly, myocytes were electrically stimulated at 0.5 Hz, and mechanical properties were recorded at a sampling rate of 240 Hz. Ca2+ transients were recorded with fluo-4 AM (Molecular Probes, Eugene, OR) while sampling at 1,000 Hz.
As previously reported (10), the indexes used to describe isotonic shortening and relengthening include peak fractional shortening (PS; peak shortening amplitude normalized to resting sarcomere length), time to peak twitch (TPT; measured between 10% and 90% above baseline), and area under the shortening (contraction) phase normalized to peak shortening amplitude (AC/PK) (Fig. 1A). The indexes for isotonic relengthening were time to relengthening (TR; measured between 10% and 90% below peak) and area under the relengthening (relaxation) phase normalized to peak amplitude (AR/PK). The indexes used to describe Ca2+ transients are summarized in Fig. 2A. The total area under the Ca2+ transient normalized to peak fluorescence (AT/PK) was used instead of separating systole and diastole. The ratio of peak fluorescence (F) to basal fluorescence (F0) was used as an index of the change in cytosolic Ca2+ during contraction, since this is independent of the intracellular concentration of the fluoroprobe (38).
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10 steady-state twitches for each myocyte. Myocytes were chosen randomly from those meeting the inclusion criteria of sharp, regular striations with visible F0 and the ability to achieve steady-state twitches and Ca2+ transients during electrical stimulation. Steady state was defined as consistent peak amplitudes during the recording period for each cell. Western blots. Hearts were either perfused (6- and 22-wk ZDF and Sprague-Dawley groups) or freshly isolated (Control and STZ groups) and were freeze clamped. As previously described (17, 21), tissue was homogenized in T-PER (Pierce) containing 5% protease inhibitor cocktail, 40 µmol/l PUGNAc (Carbogen), 1 mmol/l sodium orthovanadate, and 20 mmol/l sodium fluoride on ice. Lysates were separated on 7% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore), and equal loading of protein was initially confirmed by Sypro Ruby staining (Bio-Rad) on the membranes. Blots were probed with the appropriate antibody in casein blocking buffer and were visualized with an enhanced chemiluminescence assay (Pierce). The signal was detected with the UVP BioChemi System (UVP) and densitometry quantified with Labworks analysis software (UVP).
HPLC analysis of UDP-GlcNAc levels.
UDP-GlcNAc levels were determined as previously described by HPLC analysis of perchloric acid extracts (21, 24) with a Partisil 10 SAX column (Whatman) and a gradient from 5 to 750 mmol/l (NH4)H2PO4 and from pH 2.8 to 3.7 (31). Because this method cannot separate UDP-GlcNAc from UDP-N-acetylgalactosamine (UDP-GalNAc), the results are presented as the sum of UDP-GlcNAc and UDP-GalNAc; however, in cardiomyocytes the ratio of UDP-GlcNAc to UDP-GalNAc is 3:1 (7).
Data analysis. Data are presented as means ± SE. Differences between experimental groups were evaluated with unpaired Student's t-test for comparisons between two groups or one-way ANOVA with Bonferroni's multiple comparison test. Statistically significant differences between groups were defined as P values <0.05.
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RESULTS |
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7 wk) has been shown to adversely affect cardiomyocyte E-C coupling (29, 33). However, at 22 wk of age both contraction and relaxation were significantly impaired in the hyperglycemic ZDF group compared with controls. The time courses of the Ca2+ transients (Fig. 2; Table 3) were consistent with the mechanical indexes; for example, at 6 wk of age, there were no differences between ZDF and Lean controls in peak Ca2+ (Fig. 2B), AT/PK (Fig. 2C), TPT, or TR (Table 3). However, at 22 wk, peak Ca2+ was attenuated (Fig. 2B) and cytosolic Ca2+ clearing was slowed (Fig. 2B) in the diabetic ZDF group compared with the normoglycemic Lean group. The attenuation in peak Ca2+ did not result in predictable changes in sarcomere shortening (Fig. 1B, Table 2).
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Effect of Type 2 diabetes on cardiac levels of UDP-GlcNAc, O-GlcNAc, and OGT. There was no significant difference in UDP-GlcNAc levels between 6-wk-old Lean and ZDF groups (86 ± 6 and 87 ± 5 nmol/g wet wt, respectively). However, at 22 wk of age UDP-GlcNAc levels were significantly increased in the ZDF group compared with the age-matched Lean group (103 ± 6 vs. 88 ± 3 nmol/g wet wt; P < 0.05). UDP-GlcNAc levels were also higher in the 22-wk ZDF group compared with the 6-wk ZDF group (P < 0.05).
Consistent with the UDP-GlcNAc levels, there was no difference in O-GlcNAc levels between 6-wk-old Lean and ZDF groups, whereas there was a significant increase in O-GlcNAc at 22 wk of age between the ZDF and Lean control groups (Fig. 3, A and B). The difference in O-GlcNAc levels between the 22-wk ZDF and Lean groups was significant also when normalized to calsequestrin as a protein loading control. In all groups the anti-O-GlcNAc antibody, CTD110.6, identified 11 separate bands, all of them >57 kDa. Densitometric analysis of the individual bands in the O-GlcNAc immunoblots from 22-wk Lean and ZDF groups (Fig. 3C) revealed that the overall increase in O-GlcNAc levels was primarily due to increased O-GlcNAc on proteins in only three bands. There was no difference in OGT expression between ZDF and Lean groups at either age (Fig. 3D).
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To determine whether the age differences in O-GlcNAc and OGT expression levels were strain specific, we also examined hearts from 6- and 22-wk-old Sprague-Dawley rats. Similar to the Lean and ZDF groups, there was a significant age-related decrease in both O-GlcNAc and OGT levels (Fig. 4). Again consistent with the Lean and ZDF groups, there was no difference in UDP-GlcNAc levels between the 6- and 22-wk groups (82.3 ± 4.3 vs. 83.6 ± 5.0 nmol/g tissue wet wt, respectively).
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40% in the STZ group, but there was no difference in OGT expression levels between groups (Fig. 5).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Although we observed similar impaired relaxation and increased UDP-GlcNAc levels in the diabetic 22-wk-old ZDF group compared with the 6-wk-old normoglycemic ZDF group, paradoxically we saw a significant decrease in overall O-GlcNAc levels as well as a decrease in OGT expression. However, when comparing either 22-wk ZDF and Lean groups or 6- and 22-wk ZDF groups, we found that, independent of age and leptin levels, hyperglycemia was associated with increased O-GlcNAc levels in proteins in the very high molecular mass range (i.e., 205 kDa). This suggests that increased O-GlcNAc levels in this group of proteins may contribute to the impaired cardiomyocyte function associated with diabetes. These data suggest, therefore, that a global increase in O-GlcNAc levels is not a requirement for impaired cardiomyocyte relaxation seen with diabetes, but rather that increased O-GlcNAc on specific proteins may be responsible. This is consistent with a recent study in neonatal cardiomyocytes in which acute hyperglycemic incubation did not significantly increase overall O-GlcNAc levels but did increase O-GlcNAc levels in specific bands (4). Nevertheless, the observation that age and hyperglycemia lead to differential effects on O-GlcNAc levels on different groups of proteins is noteworthy and in need of further investigation.
Hu et al. reported that after 2–3 wk of STZ-induced diabetes there was a 20–30% increase in both OGT mRNA and protein (14), and they previously reported impaired cardiomyocyte relaxation in the same model (7), consistent with our earlier work (27). They also reported an increase in O-GlcNAc in hearts from a Type 2 model of polygenic diabetic mice, although they did not determine whether this was associated with changes in OGT expression and there were no reports of cardiomyocyte function from this Type 2 diabetic model (14). However, impaired E-C coupling has been previously reported in cardiomyocytes isolated from both ob/ob (16) and db/db (25) Type 2 diabetic mice. Consistent with the report by Hu et al., we saw an increase in overall O-GlcNAc levels in hearts from diabetic rats; however, we found no increase in OGT expression. It is not known whether insulin regulates OGT expression; thus the higher insulin levels in ZDF rats compared with the longer-term STZ model (14) could, in principle, account for the differences between our results in ZDF rats and those previously reported (14). However, we also found that in the short-term STZ model O-GlcNAc levels were increased, in the absence of any change in OGT expression levels, suggesting that increased OGT expression is not required for an increase in O-GlcNAc levels. In the 22-wk-old diabetic ZDF group, UDP-GlcNAc levels were significantly increased compared with their age-matched lean counterparts, which would suggest that increased flux through the HBP is a contributing factor in the increase in O-GlcNAc levels in this model.
An increase in HBP flux seen with diabetes may be a result not only of elevated plasma glucose levels but also of the redirection of the glucose fluxes due to increased circulating lipids and decreased activity of pyruvate dehydrogenase, both hallmarks of diabetes. It has been shown in skeletal muscle cells that increased fatty acid concentrations are associated with increased UDP-GlcNAc concentrations and expression of glucose fructose-6-phosphate amidotransferase (GFAT) (37). The latter regulates the entry of glucose into the HBP. Unfortunately, there is no information about the effect of diabetes on GFAT activity or expression in the heart; however, in skeletal muscle of non-insulin-dependent diabetes mellitus patients (41) and ob/ob mice (3) GFAT activity was increased. The lack of commercially available antibodies against GFAT precluded measurements of GFAT expression in this study; however, in the short-term STZ-treated rats no increase in UDP-GlcNAc was observed and yet O-GlcNAc levels were elevated. Thus other factors, such as increased enzyme activity, may also contribute to the increase in O-GlcNAc seen in these models. In addition, O-GlcNAc frequently competes for the same Ser/Thr residues as O-phosphorylation; thus another contributing factor to the increased O-GlcNAc levels could be a reduction in protein phosphorylation, thereby increasing the available sites for O-GlcNAc. Conversely, it is also conceivable that decreased phosphorylation may be a result of increased O-GlcNAc levels. An increase in O-GlcNAc may also occur in response to decreased O-GlcNAcase expression or activity. However, an increase in O-GlcNAcase mRNA in the heart has been reported in STZ-induced diabetes (14). The lack of a readily available source (commercial or otherwise) for an O-GlcNAcase antibody precluded the measurement of O-GlcNAcase protein expression in this study.
The fact that we did not see an increase in OGT expression in the STZ group is in contrast to the finding by Hu et al. (14). This discrepancy could be a consequence of different durations of STZ-induced diabetes (3 wk compared with 5 days here) or different species (mouse vs. rat). However, as noted above we also found no change in OGT expression between 22-wk ZDF and Lean control groups, even though the ZDF animals had been diabetic for 3 mo. To control for differences in protein loading we used calsequestrin, which exhibited no change in expression levels between Lean and ZDF groups of either age or between control and STZ-diabetic groups. Since we found no change in OGT expression in age-matched diabetic groups, regardless of whether or not the data were normalized to calsequestrin, we have a high degree of confidence in concluding that, after either a brief period of STZ-induced diabetes in Sprague-Dawley rats or a longer period of Type 2 diabetes in ZDF rats, there is no increase in OGT expression. Thus, in contrast to earlier reports (14), the increase in O-GlcNAc levels in both models of diabetes examined here cannot be attributed to altered OGT expression.
The development of obesity and diabetes in the ZDF group is due to a specific leptin receptor defect, which leads to elevated serum leptin levels. Since leptin itself has been shown to impair cardiomyocyte function (22), it is possible that the differences seen between the 22-wk-old Lean and ZDF groups could be a consequence of increased leptin levels in the ZDF group rather than the development of diabetes. However, E-C coupling was normal in the normoglycemic 6-wk ZDF group even though leptin levels were elevated at this age. Thus in ZDF rats impaired cardiomyocyte function is clearly associated with the development of hyperglycemia and is likely independent of serum leptin levels. To our knowledge, the effects on E-C coupling have not been previously described in 22-wk-old ZDF rats, and the cellular mechanisms contributing to impaired myocyte function remain to be delineated. On the basis of previous studies, it might be expected that increased O-GlcNAc levels would result in decreased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) protein expression (7, 14), thus contributing to slowed relaxation (Fig. 1D). However, we previously showed (9, 10) that diet-induced insulin resistance (9–10 wk) leads to slower cardiomyocyte relaxation and impaired SERCA function without changes in SERCA protein expression. Although we did not determine SERCA expression in this study, Golfman et al. (13) reported that there was no difference in SERCA mRNA levels between 12-wk-old ZDF rats and age-matched lean littermates. This is also consistent with studies by Belke et al. (1) and Li et al. (16), who reported that despite impaired SR function there was no difference in SERCA protein levels in hearts from db/db diabetic mice compared with nondiabetic controls. Whether SERCA function is dynamically regulated by posttranslational modification by O-GlcNAc remains to be determined.
Shifts in myosin isozyme distribution have also been implicated in diabetic cardiomyopathy, and a recent report suggests that the levels of O-GlcNAc is greater in slow skeletal muscle fibers than in fast fibers (6). In rodents, Type 1 diabetes induces a shift in myosin heavy chain (MHC) isoform distributions (i.e., reducing the fast 
-MHC and increasing the slow 
-MHC), which is consistent with myocardial contractile dysfunction (8). In the ZDF model of Type 2 diabetes there was no change in -MHC expression, although there was a significant increase in expression of -MHC (13). In db/db mice there was also an increase in -MHC, concomitant with a decrease in -MHC expression (16). It is intriguing to speculate that MHC isoform expression (or function) may be regulated by O-GlcNAc, but this remains to be determined.
Surprisingly, despite the marked hyperglycemia in the 22-wk group and increased cardiac UDP-GlcNAc concentrations, there was a significant decrease in total O-GlcNAc compared with the 6-wk group, which was associated with reduced OGT expression. Even though there was an overall decrease in O-GlcNAc levels between 6- and 22-wk ZDF groups, there was a significant increase in O-GlcNAc levels in proteins in the high molecular mass range (i.e., >205 kDa) (Fig. 6B). Proteins in the same molecular mass range also showed increased O-GlcNAc levels in the 22-wk ZDF group compared with the age-matched Lean group (Fig. 3C). It is tempting to suggest, therefore, that increased O-GlcNAc levels in this group of proteins may play a role in the development of impaired cardiomyocyte function associated with diabetes. Cardiomyocytes contain many high-molecular-mass proteins including cytoskeletal and contractile proteins such as titin, dystrophin, myosin (mentioned above), as well as proteins that may play a critical role in regulating the changes in metabolism seen in response to diabetes, such as acetyl-CoA carboxylase 2. However, standard SDS-PAGE is not well suited for separation of proteins in excess of 200 kDa; furthermore, some large proteins such as myosin tend to form aggregates under the typical conditions used for gel electrophoresis (23). To identify these proteins, further studies using protein separation techniques specifically developed for high-molecular-mass proteins (23) are needed.
It is noteworthy that a decrease in overall O-GlcNAc levels was also evident in the 6- and 22-wk-old Lean groups as well as 6- and 22-wk-old Sprague-Dawley rats, suggesting that this difference was not strain specific or dependent on the development of diabetes. Furthermore, since calsequestrin levels did not change between 6 and 22 wk of age, the reduced OGT expression that occurred between 6 and 22 wk of age cannot be attributed to a nonspecific decline in protein content over this age range. To our knowledge this is the first report of an effect of age on OGT and O-GlcNAc levels in the heart. Others have shown age-dependent changes in O-GlcNAc levels in the brain, showing an increase between 3- and 5-mo-old animals followed by a decrease over the following 2 mo (30). In the brain a reduction in O-GlcNAc levels has been associated with neurodegenerative diseases and loss of key proteins (15, 39, 40), which is consistent with suggestions that O-GlcNAc levels play a role in regulation of the ubiquitin-proteasome system (18, 42). However, it is premature to conclude that the reduction of O-GlcNAc levels in the heart seen here is necessarily detrimental given that these changes may simply reflect maturation. Clearly, further studies are warranted to determine whether cardiac O-GlcNAc levels are further reduced in older rats, which have been reported to be more susceptible to ischemic injury (19).
In conclusion, we have demonstrated for the first time that in ZDF rats the development of impaired E-C coupling is associated with the development of frank diabetes (i.e., hyperglycemia) and appears to be independent of leptin levels. Furthermore, impaired cardiomyocyte function was also associated with an increase in O-GlcNAc levels in high-molecular-mass proteins. In contrast with earlier studies (14), this increase in O-GlcNAc was associated with an increase in UDP-GlcNAc but with no concomitant increase in OGT expression. Surprisingly, there was a marked decrease in total O-GlcNAc levels between 6 and 22 wk of age in Lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. Thus the regulation of O-GlcNAc levels in the heart is clearly complex, since changes in overall O-GlcNAcylation can occur with or without changes in either OGT expression or UDP-GlcNAc levels. This complexity is further emphasized by the fact that O-GlcNAc levels on proteins in a specific molecular mass range can change in the opposite direction to overall O-GlcNAc levels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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P < 0.05 vs. 6 wk (2-way ANOVA with Bonferroni post hoc test).
