Glucosamine-Induced Endoplasmic Reticulum Dysfunction Is Associated With Accelerated Atherosclerosis in a Hyperglycemic Mouse Model
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糖尿病学杂志 2006年第1期
1 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada
3 Henderson Research Centre, McMaster University, Hamilton, Ontario, Canada
AGE, advance glycation end product; apoE, apolipoprotein E; CHOP, C/EBP (CCAAT/enhancer-binding protein) homologous protein; ER, endoplasmic reticulum; GADD153, growth arresteCand DNA damageeCinducible gene 153; GRP78, 78-kDa glucose-regulated protein; HASMC, human aortic smooth muscle cell; O-GlcNAc, O-linked N-acetylglucosamine; NF-B, nuclear factor-B; PERK, PRK (RNA-dependent protein kinase)-like ER kinase; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate; RAGE, receptor for AGEs; SREBP, sterol regulatory elementeCbinding protein; STZ, streptozotocin
ABSTRACT
Diabetes is a major independent risk factor for cardiovascular disease and stroke; however, the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. Our previous studies demonstrated that endoplasmic reticulum (ER) stresseCinducing agents, including homocysteine, promote lipid accumulation and activate inflammatory pathways—the hallmark features of atherosclerosis. We hypothesize that the accumulation of intracellular glucosamine observed in diabetes may also promote atherogenesis via a mechanism that involves ER stress. In support of this theory, we demonstrate that glucosamine can induce ER stress in cell types relevant to the development of atherosclerosis, including human aortic smooth muscle cells, monocytes, and hepatocytes. Furthermore, we show that glucosamine-induced ER stress dysregulates lipid metabolism, leading to the accumulation of cholesterol in cultured cells. To examine the relevance of the ER stress pathway in vivo, we used a streptozotocin-induced hyperglycemic apolipoprotein EeCdeficient mouse model of atherosclerosis. Using molecular biological and histological techniques, we show that hyperglycemia is associated with tissue-specific ER stress, hepatic steatosis, and accelerated atherosclerosis. This novel mechanism may not only explain how diabetes and hyperglycemia promote atherosclerosis, but also provide a potential new target for therapeutic intervention.
Type 1 and type 2 diabetes are powerful and independent risk factors for cardiovascular disease, stroke, and peripheral arterial disease. There is a strong correlation between chronic hyperglycemia and both micro- and macrovascular disease (1,2). However, despite a great deal of research, the molecular and cellular mechanisms by which chronic hyperglycemia promotes the development and/or progression of atherosclerosis are not fully understood.
The most well-defined mechanism linking hyperglycemia and downstream proatherogenic effects involves the accumulation of advance glycation end products (AGEs). AGE formation, which occurs naturally with aging, is accelerated under conditions of hyperglycemia and oxidative stress (3). AGEs induce intracellular inflammatory and oxidative stress pathways through interactions with receptors for AGEs (RAGEs) (4). Protein kinase C can be activated by AGE-RAGE interactions, resulting in proatherogenic consequences, including disruption of vascular structure and response as well as enhanced inflammatory gene expression (5,6). However, the apparent inability of antioxidant supplementation to reduce cardiovascular events in diabetic patients suggests that alternative mechanisms of hyperglycemia-induced atherosclerosis may also contribute to the progression of atherosclerosis (7eC10).
One such alternative mechanism involves the conversion of excess intracellular glucose into glucosamine by way of the hexosamine pathway. Enhanced hexosamine pathway activity has been implicated in insulin resistance, -cell death, and atherosclerosis (11eC14). One characteristic of glucosamine that has been overlooked with respect to diabetes and atherogenesis is its ability to promote the misfolding of proteins in the endoplasmic reticulum (ER), a condition defined as ER stress (15eC17). We have recently shown that activation of the cellular ER stress response correlates with atherogenic lesion development in hyperhomocysteinemic apolipoprotein E (apoE)-deficient mice (18). This finding suggests that agents and conditions that cause the misfolding of proteins in the ER may promote atherosclerosis.
In this study, using a combination of in vitro and in vivo systems, we present evidence suggesting that glucosamine-induced ER dysfunction may play a role in hepatic steatosis and accelerated atherosclerosis associated with diabetes.
RESEARCH DESIGN AND METHODS
Human hepatocarcinoma cells (HepG2) were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) containing 10% fetal bovine serum. Human monocytic cells (Thp-1) were cultured in RPMI 1640 media (Life Technologies) and 1% fetal bovine serum. Human aortic smooth muscle cells (HASMC) were cultured in Media 231 and smooth muscle growth supplement (Cascade Biologics). All cells were maintained in media adjusted to 5 mmol/l glucose in a humidified incubator at 37°C with 5% CO2. Glucose, mannitol, glucosamine, tunicamycin, A23187, and filipin were purchased from Sigma. All compounds were prepared fresh in culture medium, sterilized by filtration, and added to the cell cultures.
Mouse model.
We placed 5-week-old apoE-deficient (B6.129P2-ApoEtm1Unc) mice on a standard chow diet (TD92078; Harlan Teklad) and then randomly divided them into two groups. Mice were injected intraperitoneally over 5 consecutive days with either 40 mg/kg streptozotocin (STZ; Sigma) or an equal volume of saline (19,20). Injections were repeated at 7 weeks of age. Sustained-release insulin (0.1 unit/day per implant) or control (palmitic acid) implants (LinShin Canada) were inserted subcutaneously at the time of the first injection. Implants were replaced after 30 days, as directed by the manufacturer. Mice were killed at 15 weeks of age, and plasma and tissues were collected for further analysis. The McMaster University Animal Research Ethics Board approved all procedures.
Plasma analysis.
Whole-blood glucose levels were measured using a DEX glucometer (Bayer). Plasma glucose and lipid levels were determined in nonfasted mice, using colorimetric diagnostic kits for total cholesterol, triglycerides, and glucose purchased from Thermal DMA. Plasma lipoproteins were separated by fast protein liquid chromatography, and total cholesterol was measured in each fraction, as described previously (21).
Free-cholesterol staining.
Cells were challenged with 5 mmol/l glucosamine, 10 e蘭ol/l A23187, or 2 e蘥/ml tunicamycin for 24 h. Cells were washed three times with medium 1 (150 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl2, 20 mmol/l HEPES, pH 7.4, 2 g/l glucose), fixed with 3% paraformaldehyde, and then incubated for 2 h at room temperature with 50 e蘥/ml filipin in medium 1. Stained cells were again washed three times with medium 1, and then filipin-free cholesterol complexes were visualized by fluorescence microscopy with excitation at 335eC385 nm (emission at 420 nm) (22,23). Relative fluorescence was quantified using Sigma Scan Pro software, and results were normalized to total cell area.
Northern blot analysis.
Total RNA was isolated from cultured cells using an RNeasy total RNA kit (Qiagen). RNA (10 e蘥/lane) was size-fractionated on 2.2 mol/l formaldehyde/1.2% agarose gels, transferred to -Probe GT nylon membranes (BioRad), and hybridized using radiolabeled cDNA probes, as described previously (24). Signal intensities were quantified using a Typhoon 9410 phosphoimaging system (Amersham Pharmacia Biotech). To correct for differences in gel loading, integrated optical densities were normalized to 18S RNA. The cDNA probe encoding 78-kDa glucoseeCregulated protein (GRP78)/BiP has been described previously (25).
Immunoblot analysis.
Antibodies used for immunoblotting analysis were as follows: anti-KDEL (SPA-827; StressGen Biotech), antieCgrowth arresteCand DNA damageeCinducible gene 153 (GADD153)/C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP) (sc-575; Santa Cruz Biotechnology), RL2 (Affinity Bioreagents), antieCphosphoeCPRK (RNA-dependent protein kinase)-like ER kinase (PERK) (#3191; Cell Signaling), and antieC-actin (AC-15; Sigma). Total protein lysates from cultured cells were solubilized in SDS-PAGE sample buffer and separated on SDS-polyacrylamide gels under reducing conditions, as described previously (23,24). After incubation with the appropriate primary and horseradish peroxidaseeCconjugated secondary antibodies (Life Technologies), the membranes were developed using an ECL Plus Western blotting detection system (Amersham Biosciences), and specific bands were quantified using a Typhoon 9410 imaging system.
Luciferase assay.
HepG2 cells (50% confluent) were transfected with 3 e蘥 pNF-B-luc (nuclear factor-B [NF-B]-promoted luciferase reporter gene plasmid) or pSRF-luc (serum response factoreCpromoted luciferase reporter gene plasmid) (Stratagene), using an ExGen500 transfection reagent (MBI Fermentas). After 24 h, cells were exposed to 5 mmol/l glucosamine or tunicamycin for 6 h. Cells were lysed and luciferase activity quantified, using a TD20/20 luminometer (Turner Designs). Briefly, 30 e蘬 cell lysates was mixed with 50 e蘬 of 0.3 mmol/l D-luciferin (Sigma), and relative light units were measured immediately at room temperature. Results were normalized to total protein concentration.
Immunohistochemistry and immunofluorescence.
Mice were killed, and hearts were flushed with 1 x PBS and perfusion-fixed with 10% neutral buffer formalin. After removal, hearts, including the aortic root, were cut transversely and embedded in paraffin. Aortic root sections were collected on precoated glass slides for measurement of lesion size (hematoxylin and eosin staining) or immunohistochemical staining (26). A Vectastain ABC system (Vector Laboratories) was used for immunohistochemical staining. Sections were stained with mouse primary antibodies, using appropriate biotinylated secondary antibodies, and visualized using Nova Red. The antibody against O-linked N-acetylglucosamine (O-GlcNAc), CTD110.6, was generously provided by Dr. Gerald Hart (John Hopkins University) (27). Nonspecific staining was controlled for, using a similar section and preimmune IgG. Images were captured with a charged-coupled device color video camera (Sony) and analyzed using Northern Exposure (Empix) and SigmaScan Pro software. For immunofluorescence, sections were deparaffinized and blocked with 5% normal goat serum. Sections were incubated with the primary antibodies for 1 h and then a mixture of goat anti-mouse Alexa 594 and goat anti-rabbit Alexa 488 (Molecular Probes) for 30 min. Slides were mounted with Permafluor (Fisher Scientific) and viewed using a Zeiss Axioplan microscope.
Statistical analysis.
Results are presented as the mean ± SD. The unpaired Student’s t test was used to assess differences between experimental groups and controls. Probability values of <0.05 were considered statistically significant.
RESULTS
Glucose and glucosamine induce ER stress in cell types relevant to the development of atherosclerosis.
It has been previously shown that glucosamine can disrupt ER function, cause ER stress, and promote the activation of the unfolded protein response in CHO, Xenopus A6, and L1210 leukemic cells (15eC17). We investigated the capacity of different concentrations of glucose and glucosamine to cause an ER stress response in human cell types that are relevant to atherogenesis, including HASMC and Thp-1, as well as HepG2. The induction of mRNA encoding the diagnostic unfolded protein response marker GRP78/BiP was examined by Northern blot analysis after exposure to elevated concentrations of glucose (10 and 30 mmol/l) or glucosamine (5 and 10 mmol/l) for 4 h. As a control for the osmotic effects of these treatments, cells were exposed to 5 and 30 mmol/l mannitol. Elevated concentrations of glucosamine significantly induced GRP78/BiP mRNA levels in each of the cell types tested, suggesting that the cells were responding to conditions of ER stress (Fig. 1A). Acute exposure to high concentrations of glucose (30 mmol/l) induced GRP78/BiP levels to a greater extent in HepG2 than in HASMC or Thp-1. This observation may be indicative of a more active hexosamine pathway in HepG2. Mannitol did not induce an ER stress response in any of the cell lines examined. Similarly, levels of ER stress-induced proteins, including GRP78, heat shock protein 47 (HSP47), and GADD153/CHOP, are elevated in cells exposed to 5 mmol/l glucosamine but not mannitol (see supplementary online data [available at http://diabetes.diabetesjournals.org]). The ability of 20 e蘭ol/l azaserine, a potent inhibitor of GFAT (glutamine:fructose-6-phosphate amidotransferase), to attenuate glucose, but not glucosamine-induced ER stress, suggests that elevated concentrations of glucose cause ER stress through a glucosamine intermediate (Fig. 1B). In addition to promoting ER stress, glucosamine can promote the O-GlcNAc modification of intracellular proteins including the nuclear pore protein p62 (Fig. 1C) (28). O-GlcNAc levels can also be increased by treatment of cells with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), an inhibitor of O-GlcNAcase, the enzyme that catalyzes the removal of O-GlcNAc (29). PUGNAc treatment does not, however, promote ER stress. This result suggests that the disruption of ER homeostasis is caused by free and not protein-bound glucosamine.
Glucosamine-induced ER stress can promote lipid accumulation in cultured cells.
Previous reports, from our laboratory and others, have shown that ER stresseCinducing agents dysregulate lipid metabolism by activating the transcription factor sterol regulatory elementeCbinding protein (SREBP) (23,24,30). To determine whether glucosamine-induced ER stress can promote the dysregulation of lipid metabolism, leading to the accumulation of cholesterol, HASMC and HepG2 cells were treated with glucosamine or the classical ER stresseCinducing agents A23187 and tunicamycin. After 18eC24 h, cells were fixed in paraformaldehyde and then stained with filipin, a fluorescent dye that specifically interacts with free cholesterol (Fig. 2) (22). Fluorescence intensity was quantified and plotted against control cells that were stained with filipin in a similar way. Results indicate that glucosamine and other ER stresseCinducing agents can increase intracellular levels of free cholesterol in HepG2 and HASMC. The free cholesterol appears to accumulate around the nucleus, consistent with ER localization.
Glucosamine-induced ER stress activates NF-B in cultured cells.
It has previously been reported that ER stress inducing agents can activate the transcription factor NF-B (31). We monitored the affect of glucosamine and tunicamycin on NFB-luc (luciferase reporter construct containing an NF-B promoter element), using a transient transfection system in HepG2 cells. Our results indicate that both ER stresseCinducing agents significantly increase gene expression through the NF-B promoter (Fig. 3). In a parallel experiment, the ER stresseCinducing agents did not enhance the expression of a luciferase reporter gene containing a serum response factor promoter sequence in place of the NF-B promoter element.
STZ-induced hyperglycemia accelerates atherogenesis in an apoE-deficient mouse model.
Female apoE-deficient mice were subjected to multiple low-dose intraperitoneal injections of STZ. As a control, sustained-release insulin implants were inserted in a subgroup of STZ-injected mice. Plasma glucose levels were checked after the last STZ injection (data not shown) and were significantly higher than controls at the time of death (Table 1). Triglyceride and total cholesterol levels in 15-week-old hyperglycemic mice tended to be higher than in control mice; however, the differences were not statistically significant. The large standard deviation in total plasma cholesterol concentration in the hyperglycemic mice is most likely an early indication of the dyslipidemia that is observed in 20-week-old mice. Lipid profiles were also very similar in control and diabetic 15-week-old mice. Significant differences in total cholesterol and lipid profiles were observed in 20-week-old diabetic mice (Table 1 and Fig. 4). Specifically, VLDL and intermediate-density lipoprotein/LDL levels were elevated in 20-week-old hyperglycemic mice, a finding that is consistent with previous reports (32). At 15 weeks of age, the hyperglycemic mice had significantly advanced atherosclerosis relative to controls (Fig. 4). Mice with the slow-release insulin implant had significantly smaller atherosclerotic lesions.
Elevated O-GlcNAc in tissues from STZ-induced hyperglycemic apoE-deficient mice.
To estimate the relative intracellular concentration of glucosamine, we monitored the extent of O-GlcNAc modification of cellular proteins by immunostaining techniques, as previously described (28). Immunohistochemical and immunoblot analysis of liver, aorta, skeletal muscle, and epidydimal fat pad, using an antibody directed against O-GlcNAc (CTD110.6), indicates increased levels of intracellular glucosamine in STZ-injected hyperglycemic mice compared with controls (Fig. 5). Treatment of hyperglycemic mice with subcutaneously inserted sustained-release insulin implants (0.1 unit/day) appeared to reduce intracellular glucosamine levels in liver and aorta but increase O-linked glycosylation in the adipocytes of the fat pad. This is likely a result of the insulin redirecting glucose to insulin-sensitive tissues.
Hyperglycemia correlates with lipid accumulation and ER stress in atherosclerotic plaques and liver of hyperglycemic mice.
Various tissues from control and diabetic apoE-deficient mice were examined to determine whether there is a correlation between intracellular glucosamine levels, ER stress, and complications of diabetes, including hepatic steatosis and atherogenesis. Liver tissue from hyperglycemic mice showed lipid accumulation and increased staining for ER stress markers in hepatocytes (Fig. 6A). This result is consistent with our findings that HepG2 cells treated with glucosamine and other ER stresseCinducing agents show significant cholesterol accumulation. The intensity of both the KDEL and Oil Red O staining appeared to be lower in the insulin-treated hyperglycemic mice.
Atherosclerotic lesions in the diabetic mice stained more intensely for the ER stress markers phospho-PERK and KDEL, especially in intimal macrophages and macrophage foam cells (Fig. 6B). A comparison of lesions from 15- and 20-week-old mice suggests that ER stress levels are independent of lesion size (data not shown). Insulin treatment appeared to decrease the intensity of staining for ER stress markers in the hyperglycemic mice, a finding consistent with reduced intracellular glucosamine levels observed in Fig. 5. Coimmunofluorescence using an antibody against O-GlcNAc and antieCphospho-PERK indicates that there is a strong colocalization of markers of intracellular glucosamine and ER stress in the atherosclerotic lesions of hyperglycemic apoE-deficient mice (Fig. 6C). This observation supports our overall hypothesis that glucosamine-induced ER stress promotes accelerated atherogenesis.
DISCUSSION
A great deal of research has gone into the investigation of molecular and cellular mechanisms by which hyperglycemia may promote the development of atherosclerosis. Much of this has focused on the interaction of AGE with the receptor RAGE, which has been shown to trigger the production of intracellular reactive oxidative species and initiate inflammatory pathways. Evidence supporting accelerated atherosclerosis through AGE-induced inflammation has been obtained in a variety of experimental systems (33,34). However, the role of AGE in atherogenesis is confounded by the apparent inability of antioxidant supplementation to improve cardiovascular risk in diabetic patients, as demonstrated by several large well-controlled clinical trials (7eC10). This paradox supports the possibility that other AGE-independent mechanisms and pathways may play an important role in the atherogenic process. Here, we present evidence for a novel mechanism by which the accumulation of intracellular glucosamine, observed in hyperglycemic conditions, induces ER stress in cell types relevant to the development of atherogenesis. The cellular reaction to ER stress initiates a multifaceted, cell-specific response that can include the dysregulation of lipid metabolism and inflammation, ultimately resulting in accelerated atherosclerosis and hepatic steatosis (18,23,24).
Under physiological conditions, only 1eC3% of intracellular glucose enters the hexosamine pathway; however, flux increases with glucose concentration (11,12). Increased hexosamine pathway activity and corresponding elevated glucosamine levels have been implicated in several diabetes-associated complications, including insulin resistance (12), pancreatic -cell death (13), and atherosclerosis (14). It is not clear how intracellular glucosamine promotes cellular dysfunction, but most research has focused on the O-linked glycosylation of serine and threonine residues of specific proteins, including transcription factors, nuclear pore proteins, and signaling factors (28,35,36).
An additional intracellular effect of glucosamine that has not been investigated in the context of diabetes and atherosclerosis is its ability to promote the accumulation of unfolded proteins in the ER, a condition defined as ER stress (17). In mammals, ER stress triggers the activation of the unfolded protein response, involving three distinct integral ER membrane proteins, designated PERK, Ire1 (inositol-requiring ER-to-nucleus signal kinase 1), and ATF6 (activating transcription factor 6) (rev. in 37). Together, these proteins signal the general inhibition of protein expression and the specific induction of ER-resident chaperone expression, including GRP78/BiP (37). The ER stress response also induces expression of the transcription factor GADD153/CHOP, which is known to play a role in ER stress-induced growth arrest and programmed cell death (38). The balance of protective and proapoptotic signals triggered by ER dysfunction determines the ultimate fate of a cell. We have demonstrated that both glucose and glucosamine can promote ER stress in cultured hepatocytes, monocytes, and smooth muscle cells. In support of our in vitro findings, ER stress (39), alterations in ER morphology (17), and disruptions in the ER trafficking of specific proteins (40) have been previously observed in cultured cells exposed to glucosamine and in hyperglycemic mice. Our data suggests that the ability of glucosamine to induce ER stress is independent of increased O-linked glycosylation. This finding is significant because it supports a role for hyperglycemia-associated, glucosamine-induced ER stress that is distinct from previously identified effects of O-linked protein glycosylation, which include insulin resistance (41,42).
In recent years ER stress has been shown to affect lipid metabolism through the activation and dysregulation of the SREBPs (24,30,43). SREBPs regulate the expression of enzymes required for cholesterol and fatty acid biosynthesis and uptake (44). ER stresseCinducing agents have also been shown to activate NF-B, the transcription factor that regulates expression of inflammatory proteins (31). Together, inflammation and lipid accumulation represent the predominant characteristics of atherosclerosis. Li et al. (45) have recently shown that the loading of cultured mouse peritoneal macrophages with free cholesterol can cause ER stress that subsequently plays an essential role in the induction of inflammatory pathways. Together with our results, this finding suggests that ER stress and lipid accumulation may work through mutually reinforcing pathways that ultimately give rise to the inflammatory properties of lipid-engorged macrophages.
We have previously shown that hyperhomocysteinemia, a recognized independent risk factor for atherosclerosis, can induce ER stress in HASMCs and hepatocytes. Homocysteine-induced ER stress is associated with the dysregulation of lipid metabolism through the unregulated activation of SREBPs (24) and the induction of proapoptotic pathways (46). Hyperhomocysteinemic mice develop accelerated atherosclerosis, with lesions that show indications of elevated levels of ER stress markers, including phospho-PERK and GRP78 (18). Thus, elevated diagnostic markers of ER stress are a common characteristic of two distinctly different models of accelerated atherosclerosis involving diabetes and hyperhomocysteinemia. This observation suggests that the ER stress pathway may represent a common or unifying mechanism of atherogenesis. In support of this hypothesis, obesity, an independent risk factor for atherosclerosis, has recently been shown to promote ER stress in cell culture and mouse model systems (47). It is important to note that although hyperglycemia and hyperhomocysteinemia accelerate atherogenesis in apoE-deficient mice, a strain predisposed to lesion development, these conditions do not cause atherosclerosis in wild-type mice (18,19). This observation suggests that factors in addition to ER stress also contribute and are required for lesion development.
The effects of hyperglycemia-associated ER dysfunction are evident in both the liver and the vascular cells of the aortic wall. Therefore, we cannot determine, at this time, the relative importance of a local dysregulation of lipid metabolism in vascular cells and a distal hepatic disruption of lipid metabolism. In 15-week-old diabetic mice, total plasma cholesterol and VLDL, intermediate-density lipoprotein, and LDL cholesterol levels, in particular, are not significantly different from age-matched nondiabetic apoE-deficient mice. This finding suggests that plasma lipid levels do not play a major role in early stages of accelerated lesion development in this model. The potential for ER stress to act locally in cells of the artery wall to promote atherosclerosis is also supported by our observation that cultured human monocytes and aortic smooth muscle cells respond to glucose/glucosamine-induced ER stress by accumulating free cholesterol. At this time we do not know whether the accumulation of lipids in vascular cells is a result of increased biosynthesis and/or increased uptake. Our previous work suggesting that the ER stress dysregulates SREBPs, transcription factors that control the expression of lipid biosynthetic enzymes as well as LDL receptors, would support a role for both pathways (24).
Identification of a role for ER stress in the development and progression of atherosclerosis is important to our understanding of the molecular mechanisms that link hyperglycemia to vascular disease. Studies are now underway to determine the vascular effects of manipulating of ER stress signaling pathways in hyperglycemic mice.
ACKNOWLEDGMENTS
Funding from the Canadian Institutes of Health Research (MOP-62910) and the Heart and Stroke Foundation of Ontario (NA5556) supported this work. G.H.W is supported by a Heart and Stroke Foundation of Canada new investigators grant.
We thank Dr. Ji Zhou and Colin Halford for expert technical advice and assistance.
FOOTNOTES
Additional information can be found in an online appendix at http://diabetes.diabetesjournals.org.
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2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada
3 Henderson Research Centre, McMaster University, Hamilton, Ontario, Canada
AGE, advance glycation end product; apoE, apolipoprotein E; CHOP, C/EBP (CCAAT/enhancer-binding protein) homologous protein; ER, endoplasmic reticulum; GADD153, growth arresteCand DNA damageeCinducible gene 153; GRP78, 78-kDa glucose-regulated protein; HASMC, human aortic smooth muscle cell; O-GlcNAc, O-linked N-acetylglucosamine; NF-B, nuclear factor-B; PERK, PRK (RNA-dependent protein kinase)-like ER kinase; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate; RAGE, receptor for AGEs; SREBP, sterol regulatory elementeCbinding protein; STZ, streptozotocin
ABSTRACT
Diabetes is a major independent risk factor for cardiovascular disease and stroke; however, the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. Our previous studies demonstrated that endoplasmic reticulum (ER) stresseCinducing agents, including homocysteine, promote lipid accumulation and activate inflammatory pathways—the hallmark features of atherosclerosis. We hypothesize that the accumulation of intracellular glucosamine observed in diabetes may also promote atherogenesis via a mechanism that involves ER stress. In support of this theory, we demonstrate that glucosamine can induce ER stress in cell types relevant to the development of atherosclerosis, including human aortic smooth muscle cells, monocytes, and hepatocytes. Furthermore, we show that glucosamine-induced ER stress dysregulates lipid metabolism, leading to the accumulation of cholesterol in cultured cells. To examine the relevance of the ER stress pathway in vivo, we used a streptozotocin-induced hyperglycemic apolipoprotein EeCdeficient mouse model of atherosclerosis. Using molecular biological and histological techniques, we show that hyperglycemia is associated with tissue-specific ER stress, hepatic steatosis, and accelerated atherosclerosis. This novel mechanism may not only explain how diabetes and hyperglycemia promote atherosclerosis, but also provide a potential new target for therapeutic intervention.
Type 1 and type 2 diabetes are powerful and independent risk factors for cardiovascular disease, stroke, and peripheral arterial disease. There is a strong correlation between chronic hyperglycemia and both micro- and macrovascular disease (1,2). However, despite a great deal of research, the molecular and cellular mechanisms by which chronic hyperglycemia promotes the development and/or progression of atherosclerosis are not fully understood.
The most well-defined mechanism linking hyperglycemia and downstream proatherogenic effects involves the accumulation of advance glycation end products (AGEs). AGE formation, which occurs naturally with aging, is accelerated under conditions of hyperglycemia and oxidative stress (3). AGEs induce intracellular inflammatory and oxidative stress pathways through interactions with receptors for AGEs (RAGEs) (4). Protein kinase C can be activated by AGE-RAGE interactions, resulting in proatherogenic consequences, including disruption of vascular structure and response as well as enhanced inflammatory gene expression (5,6). However, the apparent inability of antioxidant supplementation to reduce cardiovascular events in diabetic patients suggests that alternative mechanisms of hyperglycemia-induced atherosclerosis may also contribute to the progression of atherosclerosis (7eC10).
One such alternative mechanism involves the conversion of excess intracellular glucose into glucosamine by way of the hexosamine pathway. Enhanced hexosamine pathway activity has been implicated in insulin resistance, -cell death, and atherosclerosis (11eC14). One characteristic of glucosamine that has been overlooked with respect to diabetes and atherogenesis is its ability to promote the misfolding of proteins in the endoplasmic reticulum (ER), a condition defined as ER stress (15eC17). We have recently shown that activation of the cellular ER stress response correlates with atherogenic lesion development in hyperhomocysteinemic apolipoprotein E (apoE)-deficient mice (18). This finding suggests that agents and conditions that cause the misfolding of proteins in the ER may promote atherosclerosis.
In this study, using a combination of in vitro and in vivo systems, we present evidence suggesting that glucosamine-induced ER dysfunction may play a role in hepatic steatosis and accelerated atherosclerosis associated with diabetes.
RESEARCH DESIGN AND METHODS
Human hepatocarcinoma cells (HepG2) were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) containing 10% fetal bovine serum. Human monocytic cells (Thp-1) were cultured in RPMI 1640 media (Life Technologies) and 1% fetal bovine serum. Human aortic smooth muscle cells (HASMC) were cultured in Media 231 and smooth muscle growth supplement (Cascade Biologics). All cells were maintained in media adjusted to 5 mmol/l glucose in a humidified incubator at 37°C with 5% CO2. Glucose, mannitol, glucosamine, tunicamycin, A23187, and filipin were purchased from Sigma. All compounds were prepared fresh in culture medium, sterilized by filtration, and added to the cell cultures.
Mouse model.
We placed 5-week-old apoE-deficient (B6.129P2-ApoEtm1Unc) mice on a standard chow diet (TD92078; Harlan Teklad) and then randomly divided them into two groups. Mice were injected intraperitoneally over 5 consecutive days with either 40 mg/kg streptozotocin (STZ; Sigma) or an equal volume of saline (19,20). Injections were repeated at 7 weeks of age. Sustained-release insulin (0.1 unit/day per implant) or control (palmitic acid) implants (LinShin Canada) were inserted subcutaneously at the time of the first injection. Implants were replaced after 30 days, as directed by the manufacturer. Mice were killed at 15 weeks of age, and plasma and tissues were collected for further analysis. The McMaster University Animal Research Ethics Board approved all procedures.
Plasma analysis.
Whole-blood glucose levels were measured using a DEX glucometer (Bayer). Plasma glucose and lipid levels were determined in nonfasted mice, using colorimetric diagnostic kits for total cholesterol, triglycerides, and glucose purchased from Thermal DMA. Plasma lipoproteins were separated by fast protein liquid chromatography, and total cholesterol was measured in each fraction, as described previously (21).
Free-cholesterol staining.
Cells were challenged with 5 mmol/l glucosamine, 10 e蘭ol/l A23187, or 2 e蘥/ml tunicamycin for 24 h. Cells were washed three times with medium 1 (150 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl2, 20 mmol/l HEPES, pH 7.4, 2 g/l glucose), fixed with 3% paraformaldehyde, and then incubated for 2 h at room temperature with 50 e蘥/ml filipin in medium 1. Stained cells were again washed three times with medium 1, and then filipin-free cholesterol complexes were visualized by fluorescence microscopy with excitation at 335eC385 nm (emission at 420 nm) (22,23). Relative fluorescence was quantified using Sigma Scan Pro software, and results were normalized to total cell area.
Northern blot analysis.
Total RNA was isolated from cultured cells using an RNeasy total RNA kit (Qiagen). RNA (10 e蘥/lane) was size-fractionated on 2.2 mol/l formaldehyde/1.2% agarose gels, transferred to -Probe GT nylon membranes (BioRad), and hybridized using radiolabeled cDNA probes, as described previously (24). Signal intensities were quantified using a Typhoon 9410 phosphoimaging system (Amersham Pharmacia Biotech). To correct for differences in gel loading, integrated optical densities were normalized to 18S RNA. The cDNA probe encoding 78-kDa glucoseeCregulated protein (GRP78)/BiP has been described previously (25).
Immunoblot analysis.
Antibodies used for immunoblotting analysis were as follows: anti-KDEL (SPA-827; StressGen Biotech), antieCgrowth arresteCand DNA damageeCinducible gene 153 (GADD153)/C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP) (sc-575; Santa Cruz Biotechnology), RL2 (Affinity Bioreagents), antieCphosphoeCPRK (RNA-dependent protein kinase)-like ER kinase (PERK) (#3191; Cell Signaling), and antieC-actin (AC-15; Sigma). Total protein lysates from cultured cells were solubilized in SDS-PAGE sample buffer and separated on SDS-polyacrylamide gels under reducing conditions, as described previously (23,24). After incubation with the appropriate primary and horseradish peroxidaseeCconjugated secondary antibodies (Life Technologies), the membranes were developed using an ECL Plus Western blotting detection system (Amersham Biosciences), and specific bands were quantified using a Typhoon 9410 imaging system.
Luciferase assay.
HepG2 cells (50% confluent) were transfected with 3 e蘥 pNF-B-luc (nuclear factor-B [NF-B]-promoted luciferase reporter gene plasmid) or pSRF-luc (serum response factoreCpromoted luciferase reporter gene plasmid) (Stratagene), using an ExGen500 transfection reagent (MBI Fermentas). After 24 h, cells were exposed to 5 mmol/l glucosamine or tunicamycin for 6 h. Cells were lysed and luciferase activity quantified, using a TD20/20 luminometer (Turner Designs). Briefly, 30 e蘬 cell lysates was mixed with 50 e蘬 of 0.3 mmol/l D-luciferin (Sigma), and relative light units were measured immediately at room temperature. Results were normalized to total protein concentration.
Immunohistochemistry and immunofluorescence.
Mice were killed, and hearts were flushed with 1 x PBS and perfusion-fixed with 10% neutral buffer formalin. After removal, hearts, including the aortic root, were cut transversely and embedded in paraffin. Aortic root sections were collected on precoated glass slides for measurement of lesion size (hematoxylin and eosin staining) or immunohistochemical staining (26). A Vectastain ABC system (Vector Laboratories) was used for immunohistochemical staining. Sections were stained with mouse primary antibodies, using appropriate biotinylated secondary antibodies, and visualized using Nova Red. The antibody against O-linked N-acetylglucosamine (O-GlcNAc), CTD110.6, was generously provided by Dr. Gerald Hart (John Hopkins University) (27). Nonspecific staining was controlled for, using a similar section and preimmune IgG. Images were captured with a charged-coupled device color video camera (Sony) and analyzed using Northern Exposure (Empix) and SigmaScan Pro software. For immunofluorescence, sections were deparaffinized and blocked with 5% normal goat serum. Sections were incubated with the primary antibodies for 1 h and then a mixture of goat anti-mouse Alexa 594 and goat anti-rabbit Alexa 488 (Molecular Probes) for 30 min. Slides were mounted with Permafluor (Fisher Scientific) and viewed using a Zeiss Axioplan microscope.
Statistical analysis.
Results are presented as the mean ± SD. The unpaired Student’s t test was used to assess differences between experimental groups and controls. Probability values of <0.05 were considered statistically significant.
RESULTS
Glucose and glucosamine induce ER stress in cell types relevant to the development of atherosclerosis.
It has been previously shown that glucosamine can disrupt ER function, cause ER stress, and promote the activation of the unfolded protein response in CHO, Xenopus A6, and L1210 leukemic cells (15eC17). We investigated the capacity of different concentrations of glucose and glucosamine to cause an ER stress response in human cell types that are relevant to atherogenesis, including HASMC and Thp-1, as well as HepG2. The induction of mRNA encoding the diagnostic unfolded protein response marker GRP78/BiP was examined by Northern blot analysis after exposure to elevated concentrations of glucose (10 and 30 mmol/l) or glucosamine (5 and 10 mmol/l) for 4 h. As a control for the osmotic effects of these treatments, cells were exposed to 5 and 30 mmol/l mannitol. Elevated concentrations of glucosamine significantly induced GRP78/BiP mRNA levels in each of the cell types tested, suggesting that the cells were responding to conditions of ER stress (Fig. 1A). Acute exposure to high concentrations of glucose (30 mmol/l) induced GRP78/BiP levels to a greater extent in HepG2 than in HASMC or Thp-1. This observation may be indicative of a more active hexosamine pathway in HepG2. Mannitol did not induce an ER stress response in any of the cell lines examined. Similarly, levels of ER stress-induced proteins, including GRP78, heat shock protein 47 (HSP47), and GADD153/CHOP, are elevated in cells exposed to 5 mmol/l glucosamine but not mannitol (see supplementary online data [available at http://diabetes.diabetesjournals.org]). The ability of 20 e蘭ol/l azaserine, a potent inhibitor of GFAT (glutamine:fructose-6-phosphate amidotransferase), to attenuate glucose, but not glucosamine-induced ER stress, suggests that elevated concentrations of glucose cause ER stress through a glucosamine intermediate (Fig. 1B). In addition to promoting ER stress, glucosamine can promote the O-GlcNAc modification of intracellular proteins including the nuclear pore protein p62 (Fig. 1C) (28). O-GlcNAc levels can also be increased by treatment of cells with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), an inhibitor of O-GlcNAcase, the enzyme that catalyzes the removal of O-GlcNAc (29). PUGNAc treatment does not, however, promote ER stress. This result suggests that the disruption of ER homeostasis is caused by free and not protein-bound glucosamine.
Glucosamine-induced ER stress can promote lipid accumulation in cultured cells.
Previous reports, from our laboratory and others, have shown that ER stresseCinducing agents dysregulate lipid metabolism by activating the transcription factor sterol regulatory elementeCbinding protein (SREBP) (23,24,30). To determine whether glucosamine-induced ER stress can promote the dysregulation of lipid metabolism, leading to the accumulation of cholesterol, HASMC and HepG2 cells were treated with glucosamine or the classical ER stresseCinducing agents A23187 and tunicamycin. After 18eC24 h, cells were fixed in paraformaldehyde and then stained with filipin, a fluorescent dye that specifically interacts with free cholesterol (Fig. 2) (22). Fluorescence intensity was quantified and plotted against control cells that were stained with filipin in a similar way. Results indicate that glucosamine and other ER stresseCinducing agents can increase intracellular levels of free cholesterol in HepG2 and HASMC. The free cholesterol appears to accumulate around the nucleus, consistent with ER localization.
Glucosamine-induced ER stress activates NF-B in cultured cells.
It has previously been reported that ER stress inducing agents can activate the transcription factor NF-B (31). We monitored the affect of glucosamine and tunicamycin on NFB-luc (luciferase reporter construct containing an NF-B promoter element), using a transient transfection system in HepG2 cells. Our results indicate that both ER stresseCinducing agents significantly increase gene expression through the NF-B promoter (Fig. 3). In a parallel experiment, the ER stresseCinducing agents did not enhance the expression of a luciferase reporter gene containing a serum response factor promoter sequence in place of the NF-B promoter element.
STZ-induced hyperglycemia accelerates atherogenesis in an apoE-deficient mouse model.
Female apoE-deficient mice were subjected to multiple low-dose intraperitoneal injections of STZ. As a control, sustained-release insulin implants were inserted in a subgroup of STZ-injected mice. Plasma glucose levels were checked after the last STZ injection (data not shown) and were significantly higher than controls at the time of death (Table 1). Triglyceride and total cholesterol levels in 15-week-old hyperglycemic mice tended to be higher than in control mice; however, the differences were not statistically significant. The large standard deviation in total plasma cholesterol concentration in the hyperglycemic mice is most likely an early indication of the dyslipidemia that is observed in 20-week-old mice. Lipid profiles were also very similar in control and diabetic 15-week-old mice. Significant differences in total cholesterol and lipid profiles were observed in 20-week-old diabetic mice (Table 1 and Fig. 4). Specifically, VLDL and intermediate-density lipoprotein/LDL levels were elevated in 20-week-old hyperglycemic mice, a finding that is consistent with previous reports (32). At 15 weeks of age, the hyperglycemic mice had significantly advanced atherosclerosis relative to controls (Fig. 4). Mice with the slow-release insulin implant had significantly smaller atherosclerotic lesions.
Elevated O-GlcNAc in tissues from STZ-induced hyperglycemic apoE-deficient mice.
To estimate the relative intracellular concentration of glucosamine, we monitored the extent of O-GlcNAc modification of cellular proteins by immunostaining techniques, as previously described (28). Immunohistochemical and immunoblot analysis of liver, aorta, skeletal muscle, and epidydimal fat pad, using an antibody directed against O-GlcNAc (CTD110.6), indicates increased levels of intracellular glucosamine in STZ-injected hyperglycemic mice compared with controls (Fig. 5). Treatment of hyperglycemic mice with subcutaneously inserted sustained-release insulin implants (0.1 unit/day) appeared to reduce intracellular glucosamine levels in liver and aorta but increase O-linked glycosylation in the adipocytes of the fat pad. This is likely a result of the insulin redirecting glucose to insulin-sensitive tissues.
Hyperglycemia correlates with lipid accumulation and ER stress in atherosclerotic plaques and liver of hyperglycemic mice.
Various tissues from control and diabetic apoE-deficient mice were examined to determine whether there is a correlation between intracellular glucosamine levels, ER stress, and complications of diabetes, including hepatic steatosis and atherogenesis. Liver tissue from hyperglycemic mice showed lipid accumulation and increased staining for ER stress markers in hepatocytes (Fig. 6A). This result is consistent with our findings that HepG2 cells treated with glucosamine and other ER stresseCinducing agents show significant cholesterol accumulation. The intensity of both the KDEL and Oil Red O staining appeared to be lower in the insulin-treated hyperglycemic mice.
Atherosclerotic lesions in the diabetic mice stained more intensely for the ER stress markers phospho-PERK and KDEL, especially in intimal macrophages and macrophage foam cells (Fig. 6B). A comparison of lesions from 15- and 20-week-old mice suggests that ER stress levels are independent of lesion size (data not shown). Insulin treatment appeared to decrease the intensity of staining for ER stress markers in the hyperglycemic mice, a finding consistent with reduced intracellular glucosamine levels observed in Fig. 5. Coimmunofluorescence using an antibody against O-GlcNAc and antieCphospho-PERK indicates that there is a strong colocalization of markers of intracellular glucosamine and ER stress in the atherosclerotic lesions of hyperglycemic apoE-deficient mice (Fig. 6C). This observation supports our overall hypothesis that glucosamine-induced ER stress promotes accelerated atherogenesis.
DISCUSSION
A great deal of research has gone into the investigation of molecular and cellular mechanisms by which hyperglycemia may promote the development of atherosclerosis. Much of this has focused on the interaction of AGE with the receptor RAGE, which has been shown to trigger the production of intracellular reactive oxidative species and initiate inflammatory pathways. Evidence supporting accelerated atherosclerosis through AGE-induced inflammation has been obtained in a variety of experimental systems (33,34). However, the role of AGE in atherogenesis is confounded by the apparent inability of antioxidant supplementation to improve cardiovascular risk in diabetic patients, as demonstrated by several large well-controlled clinical trials (7eC10). This paradox supports the possibility that other AGE-independent mechanisms and pathways may play an important role in the atherogenic process. Here, we present evidence for a novel mechanism by which the accumulation of intracellular glucosamine, observed in hyperglycemic conditions, induces ER stress in cell types relevant to the development of atherogenesis. The cellular reaction to ER stress initiates a multifaceted, cell-specific response that can include the dysregulation of lipid metabolism and inflammation, ultimately resulting in accelerated atherosclerosis and hepatic steatosis (18,23,24).
Under physiological conditions, only 1eC3% of intracellular glucose enters the hexosamine pathway; however, flux increases with glucose concentration (11,12). Increased hexosamine pathway activity and corresponding elevated glucosamine levels have been implicated in several diabetes-associated complications, including insulin resistance (12), pancreatic -cell death (13), and atherosclerosis (14). It is not clear how intracellular glucosamine promotes cellular dysfunction, but most research has focused on the O-linked glycosylation of serine and threonine residues of specific proteins, including transcription factors, nuclear pore proteins, and signaling factors (28,35,36).
An additional intracellular effect of glucosamine that has not been investigated in the context of diabetes and atherosclerosis is its ability to promote the accumulation of unfolded proteins in the ER, a condition defined as ER stress (17). In mammals, ER stress triggers the activation of the unfolded protein response, involving three distinct integral ER membrane proteins, designated PERK, Ire1 (inositol-requiring ER-to-nucleus signal kinase 1), and ATF6 (activating transcription factor 6) (rev. in 37). Together, these proteins signal the general inhibition of protein expression and the specific induction of ER-resident chaperone expression, including GRP78/BiP (37). The ER stress response also induces expression of the transcription factor GADD153/CHOP, which is known to play a role in ER stress-induced growth arrest and programmed cell death (38). The balance of protective and proapoptotic signals triggered by ER dysfunction determines the ultimate fate of a cell. We have demonstrated that both glucose and glucosamine can promote ER stress in cultured hepatocytes, monocytes, and smooth muscle cells. In support of our in vitro findings, ER stress (39), alterations in ER morphology (17), and disruptions in the ER trafficking of specific proteins (40) have been previously observed in cultured cells exposed to glucosamine and in hyperglycemic mice. Our data suggests that the ability of glucosamine to induce ER stress is independent of increased O-linked glycosylation. This finding is significant because it supports a role for hyperglycemia-associated, glucosamine-induced ER stress that is distinct from previously identified effects of O-linked protein glycosylation, which include insulin resistance (41,42).
In recent years ER stress has been shown to affect lipid metabolism through the activation and dysregulation of the SREBPs (24,30,43). SREBPs regulate the expression of enzymes required for cholesterol and fatty acid biosynthesis and uptake (44). ER stresseCinducing agents have also been shown to activate NF-B, the transcription factor that regulates expression of inflammatory proteins (31). Together, inflammation and lipid accumulation represent the predominant characteristics of atherosclerosis. Li et al. (45) have recently shown that the loading of cultured mouse peritoneal macrophages with free cholesterol can cause ER stress that subsequently plays an essential role in the induction of inflammatory pathways. Together with our results, this finding suggests that ER stress and lipid accumulation may work through mutually reinforcing pathways that ultimately give rise to the inflammatory properties of lipid-engorged macrophages.
We have previously shown that hyperhomocysteinemia, a recognized independent risk factor for atherosclerosis, can induce ER stress in HASMCs and hepatocytes. Homocysteine-induced ER stress is associated with the dysregulation of lipid metabolism through the unregulated activation of SREBPs (24) and the induction of proapoptotic pathways (46). Hyperhomocysteinemic mice develop accelerated atherosclerosis, with lesions that show indications of elevated levels of ER stress markers, including phospho-PERK and GRP78 (18). Thus, elevated diagnostic markers of ER stress are a common characteristic of two distinctly different models of accelerated atherosclerosis involving diabetes and hyperhomocysteinemia. This observation suggests that the ER stress pathway may represent a common or unifying mechanism of atherogenesis. In support of this hypothesis, obesity, an independent risk factor for atherosclerosis, has recently been shown to promote ER stress in cell culture and mouse model systems (47). It is important to note that although hyperglycemia and hyperhomocysteinemia accelerate atherogenesis in apoE-deficient mice, a strain predisposed to lesion development, these conditions do not cause atherosclerosis in wild-type mice (18,19). This observation suggests that factors in addition to ER stress also contribute and are required for lesion development.
The effects of hyperglycemia-associated ER dysfunction are evident in both the liver and the vascular cells of the aortic wall. Therefore, we cannot determine, at this time, the relative importance of a local dysregulation of lipid metabolism in vascular cells and a distal hepatic disruption of lipid metabolism. In 15-week-old diabetic mice, total plasma cholesterol and VLDL, intermediate-density lipoprotein, and LDL cholesterol levels, in particular, are not significantly different from age-matched nondiabetic apoE-deficient mice. This finding suggests that plasma lipid levels do not play a major role in early stages of accelerated lesion development in this model. The potential for ER stress to act locally in cells of the artery wall to promote atherosclerosis is also supported by our observation that cultured human monocytes and aortic smooth muscle cells respond to glucose/glucosamine-induced ER stress by accumulating free cholesterol. At this time we do not know whether the accumulation of lipids in vascular cells is a result of increased biosynthesis and/or increased uptake. Our previous work suggesting that the ER stress dysregulates SREBPs, transcription factors that control the expression of lipid biosynthetic enzymes as well as LDL receptors, would support a role for both pathways (24).
Identification of a role for ER stress in the development and progression of atherosclerosis is important to our understanding of the molecular mechanisms that link hyperglycemia to vascular disease. Studies are now underway to determine the vascular effects of manipulating of ER stress signaling pathways in hyperglycemic mice.
ACKNOWLEDGMENTS
Funding from the Canadian Institutes of Health Research (MOP-62910) and the Heart and Stroke Foundation of Ontario (NA5556) supported this work. G.H.W is supported by a Heart and Stroke Foundation of Canada new investigators grant.
We thank Dr. Ji Zhou and Colin Halford for expert technical advice and assistance.
FOOTNOTES
Additional information can be found in an online appendix at http://diabetes.diabetesjournals.org.
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