Original Article

Glucosamine-Induced Endoplasmic ReticulumDysfunction Is Associated With AcceleratedAtherosclerosis in a Hyperglycemic Mouse ModelGeoff H. Werstuck,

1,2,3Mohammad I. Khan,

3Giuseppe Femia,

1Anna J. Kim,

1Vivienne Tedesco,

1

Bernardo Trigatti,1

and Yuanyuan Shi3

Diabetes is a major independent risk factor for cardiovas-

cular disease and stroke; however, the molecular and cel-

lular mechanisms by which diabetes contributes to the

development of vascular disease are not fully understood.

Our previous studies demonstrated that endoplasmic retic-

ulum (ER) stress–inducing agents, including homocysteine,

promote lipid accumulation and activate inflammatory

pathways—the hallmark features of atherosclerosis. We

hypothesize that the accumulation of intracellular glu-

cosamine observed in diabetes may also promote athero-

genesis 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 me-

tabolism, 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 hyper-

glycemic apolipoprotein E–deficient mouse model of ath-

erosclerosis. 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 ex-

plain how diabetes and hyperglycemia promote atheroscle-

rosis, but also provide a potential new target for

therapeutic intervention. Diabetes 55:93–101, 2006

Type 1 and type 2 diabetes are powerful andindependent risk factors for cardiovascular dis-ease, 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, themolecular and cellular mechanisms by which chronichyperglycemia promotes the development and/or progres-sion of atherosclerosis are not fully understood.

The most well-defined mechanism linking hyperglyce-mia and downstream proatherogenic effects involves theaccumulation of advance glycation end products (AGEs).AGE formation, which occurs naturally with aging, isaccelerated under conditions of hyperglycemia and oxida-tive stress (3). AGEs induce intracellular inflamma-tory and oxidative stress pathways through interactionswith receptors for AGEs (RAGEs) (4). Protein kinase Ccan be activated by AGE-RAGE interactions, resulting inproatherogenic consequences, including disruption of vas-cular structure and response as well as enhanced inflam-matory gene expression (5,6). However, the apparentinability of antioxidant supplementation to reduce car-diovascular events in diabetic patients suggests thatalternative mechanisms of hyperglycemia-induced athero-sclerosis may also contribute to the progression of athero-sclerosis (7–10).

One such alternative mechanism involves the conver-sion of excess intracellular glucose into glucosamine byway of the hexosamine pathway. Enhanced hexosaminepathway activity has been implicated in insulin resistance,�-cell death, and atherosclerosis (11–14). One character-istic of glucosamine that has been overlooked with respectto diabetes and atherogenesis is its ability to promote themisfolding of proteins in the endoplasmic reticulum (ER),a condition defined as ER stress (15–17). We have recentlyshown that activation of the cellular ER stress responsecorrelates with atherogenic lesion development in hyper-homocysteinemic apolipoprotein E (apoE)-deficient mice(18). This finding suggests that agents and conditions thatcause the misfolding of proteins in the ER may promoteatherosclerosis.

In this study, using a combination of in vitro and in vivosystems, we present evidence suggesting that glu-cosamine-induced ER dysfunction may play a role inhepatic steatosis and accelerated atherosclerosis associ-ated with diabetes.

From the 1Department of Biochemistry and Biomedical Sciences, McMasterUniversity, Hamilton, Ontario, Canada; the 2Department of Medicine, McMas-ter University, Hamilton, Ontario, Canada; and the 3Henderson ResearchCentre, McMaster University, Hamilton, Ontario, Canada.

Address correspondence and reprint requests to Geoff H. Werstuck, Hen-derson Research Centre, 711 Concession St., Hamilton, Ontario, Canada, L8V1C3. E-mail: gwerstuck@thrombosis.hhscr.org.

Received for publication 18 May 2005 and accepted in revised form 3October 2005.

Additional information can be found in an online appendix at http://diabetes.diabetesjournals.org.

AGE, advance glycation end product; apoE, apolipoprotein E; CHOP, C/EBP(CCAAT/enhancer-binding protein) homologous protein; ER, endoplasmicreticulum; GADD153, growth arrest– and DNA damage–inducible gene 153;GRP78, 78-kDa glucose-regulated protein; HASMC, human aortic smoothmuscle 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 element–binding protein;STZ, streptozotocin.

© 2006 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

DIABETES, VOL. 55, JANUARY 2006 93

RESEARCH DESIGN AND METHODS

Human hepatocarcinoma cells (HepG2) were cultured in Dulbecco’s modifiedEagle’s medium (Life Technologies) containing 10% fetal bovine serum.Human monocytic cells (Thp-1) were cultured in RPMI 1640 media (LifeTechnologies) 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/lglucose in a humidified incubator at 37°C with 5% CO2. Glucose, mannitol,glucosamine, tunicamycin, A23187, and filipin were purchased from Sigma. Allcompounds 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 randomlydivided them into two groups. Mice were injected intraperitoneally over 5consecutive days with either 40 mg/kg streptozotocin (STZ; Sigma) or an equalvolume 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 thefirst injection. Implants were replaced after 30 days, as directed by themanufacturer. Mice were killed at 15 weeks of age, and plasma and tissueswere collected for further analysis. The McMaster University Animal ResearchEthics Board approved all procedures.Plasma analysis. Whole-blood glucose levels were measured using a DEXglucometer (Bayer). Plasma glucose and lipid levels were determined innonfasted mice, using colorimetric diagnostic kits for total cholesterol,triglycerides, and glucose purchased from Thermal DMA. Plasma lipoproteinswere separated by fast protein liquid chromatography, and total cholesterolwas measured in each fraction, as described previously (21).Free-cholesterol staining. Cells were challenged with 5 mmol/l glu-cosamine, 10 �mol/l A23187, or 2 �g/ml tunicamycin for 24 h. Cells werewashed three times with medium 1 (150 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/lCaCl2, 20 mmol/l HEPES, pH 7.4, 2 g/l glucose), fixed with 3% paraformalde-hyde, and then incubated for 2 h at room temperature with 50 �g/ml filipin inmedium 1. Stained cells were again washed three times with medium 1, andthen filipin-free cholesterol complexes were visualized by fluorescence mi-croscopy with excitation at 335–385 nm (emission at 420 nm) (22,23). Relativefluorescence was quantified using Sigma Scan Pro software, and results werenormalized to total cell area.Northern blot analysis. Total RNA was isolated from cultured cells using anRNeasy total RNA kit (Qiagen). RNA (10 �g/lane) was size-fractionated on 2.2mol/l formaldehyde/1.2% agarose gels, transferred to �-Probe GT nylon mem-branes (BioRad), and hybridized using radiolabeled cDNA probes, as de-scribed previously (24). Signal intensities were quantified using a Typhoon9410 phosphoimaging system (Amersham Pharmacia Biotech). To correct fordifferences in gel loading, integrated optical densities were normalized to 18SRNA. The cDNA probe encoding 78-kDa glucose–regulated protein (GRP78)/BiP has been described previously (25).Immunoblot analysis. Antibodies used for immunoblotting analysis were asfollows: anti-KDEL (SPA-827; StressGen Biotech), anti–growth arrest–andDNA damage–inducible gene 153 (GADD153)/C/EBP (CCAAT/enhancer-bind-ing protein) homologous protein (CHOP) (sc-575; Santa Cruz Biotechnology),RL2 (Affinity Bioreagents), anti–phospho–PRK (RNA-dependent protein ki-nase)-like ER kinase (PERK) (#3191; Cell Signaling), and anti–�-actin (AC-15;Sigma). Total protein lysates from cultured cells were solubilized in SDS-PAGE sample buffer and separated on SDS-polyacrylamide gels under reduc-ing conditions, as described previously (23,24). After incubation with theappropriate primary and horseradish peroxidase–conjugated secondary anti-bodies (Life Technologies), the membranes were developed using an ECL PlusWestern blotting detection system (Amersham Biosciences), and specificbands were quantified using a Typhoon 9410 imaging system.Luciferase assay. HepG2 cells (50% confluent) were transfected with 3 �gpNF-�B-luc (nuclear factor-�B [NF-�B]-promoted luciferase reporter geneplasmid) or pSRF-luc (serum response factor–promoted luciferase reportergene plasmid) (Stratagene), using an ExGen500 transfection reagent (MBIFermentas). After 24 h, cells were exposed to 5 mmol/l glucosamine ortunicamycin for 6 h. Cells were lysed and luciferase activity quantified, usinga TD20/20 luminometer (Turner Designs). Briefly, 30 �l cell lysates was mixedwith 50 �l of 0.3 mmol/l D-luciferin (Sigma), and relative light units weremeasured immediately at room temperature. Results were normalized to totalprotein concentration.Immunohistochemistry and immunofluorescence. Mice were killed, andhearts were flushed with 1 � PBS and perfusion-fixed with 10% neutral bufferformalin. After removal, hearts, including the aortic root, were cut trans-versely and embedded in paraffin. Aortic root sections were collected onprecoated glass slides for measurement of lesion size (hematoxylin and eosinstaining) or immunohistochemical staining (26). A Vectastain ABC system

(Vector Laboratories) was used for immunohistochemical staining. Sectionswere stained with mouse primary antibodies, using appropriate biotinylatedsecondary antibodies, and visualized using Nova Red. The antibody againstO-linked N-acetylglucosamine (O-GlcNAc), CTD110.6, was generously pro-vided by Dr. Gerald Hart (John Hopkins University) (27). Nonspecific stainingwas controlled for, using a similar section and preimmune IgG. Images werecaptured with a charged-coupled device color video camera (Sony) andanalyzed using Northern Exposure (Empix) and SigmaScan Pro software. Forimmunofluorescence, sections were deparaffinized and blocked with 5%normal goat serum. Sections were incubated with the primary antibodies for1 h and then a mixture of goat anti-mouse Alexa 594 and goat anti-rabbit Alexa488 (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 unpairedStudent’s t test was used to assess differences between experimental groupsand controls. Probability values of �0.05 were considered statistically signif-icant.

RESULTS

Glucose and glucosamine induce ER stress in celltypes relevant to the development of atherosclerosis.It has been previously shown that glucosamine can disruptER function, cause ER stress, and promote the activationof the unfolded protein response in CHO, Xenopus A6, andL1210 leukemic cells (15–17). We investigated the capacityof different concentrations of glucose and glucosamine tocause an ER stress response in human cell types that arerelevant to atherogenesis, including HASMC and Thp-1, aswell as HepG2. The induction of mRNA encoding thediagnostic unfolded protein response marker GRP78/BiPwas examined by Northern blot analysis after exposure toelevated concentrations of glucose (10 and 30 mmol/l) orglucosamine (5 and 10 mmol/l) for 4 h. As a control for theosmotic effects of these treatments, cells were exposed to5 and 30 mmol/l mannitol. Elevated concentrations ofglucosamine significantly induced GRP78/BiP mRNA lev-els in each of the cell types tested, suggesting that the cellswere responding to conditions of ER stress (Fig. 1A).Acute exposure to high concentrations of glucose (30mmol/l) induced GRP78/BiP levels to a greater extent inHepG2 than in HASMC or Thp-1. This observation may beindicative of a more active hexosamine pathway in HepG2.Mannitol did not induce an ER stress response in any ofthe cell lines examined. Similarly, levels of ER stress-induced proteins, including GRP78, heat shock protein 47(HSP47), and GADD153/CHOP, are elevated in cells ex-posed to 5 mmol/l glucosamine but not mannitol (seesupplementary online data [available at http://diabetes.diabetesjournals.org]). The ability of 20 �mol/l azaserine, apotent inhibitor of GFAT (glutamine:fructose-6-phosphateamidotransferase), to attenuate glucose, but not glu-cosamine-induced ER stress, suggests that elevated con-centrations of glucose cause ER stress through aglucosamine intermediate (Fig. 1B). In addition to promot-ing ER stress, glucosamine can promote the O-GlcNAcmodification of intracellular proteins including the nuclearpore protein p62 (Fig. 1C) (28). O-GlcNAc levels can alsobe increased by treatment of cells with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, the enzyme thatcatalyzes the removal of O-GlcNAc (29). PUGNAc treat-ment does not, however, promote ER stress. This resultsuggests that the disruption of ER homeostasis is causedby free and not protein-bound glucosamine.Glucosamine-induced ER stress can promote lipidaccumulation in cultured cells. Previous reports, fromour laboratory and others, have shown that ER stress–inducing agents dysregulate lipid metabolism by activating

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94 DIABETES, VOL. 55, JANUARY 2006

the transcription factor sterol regulatory element–bindingprotein (SREBP) (23,24,30). To determine whether glu-cosamine-induced ER stress can promote the dysregula-tion of lipid metabolism, leading to the accumulation ofcholesterol, HASMC and HepG2 cells were treated withglucosamine or the classical ER stress–inducing agentsA23187 and tunicamycin. After 18–24 h, cells were fixed inparaformaldehyde and then stained with filipin, a fluores-cent dye that specifically interacts with free cholesterol(Fig. 2) (22). Fluorescence intensity was quantified andplotted against control cells that were stained with filipinin a similar way. Results indicate that glucosamine andother ER stress–inducing agents can increase intracellularlevels of free cholesterol in HepG2 and HASMC. The freecholesterol 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 ERstress inducing agents can activate the transcription factorNF-�B (31). We monitored the affect of glucosamine andtunicamycin on NF�B-luc (luciferase reporter constructcontaining an NF-�B promoter element), using a transienttransfection system in HepG2 cells. Our results indicatethat both ER stress–inducing agents significantly increasegene expression through the NF-�B promoter (Fig. 3). In aparallel experiment, the ER stress–inducing agents did notenhance the expression of a luciferase reporter genecontaining a serum response factor promoter sequence inplace of the NF-�B promoter element.

STZ-induced hyperglycemia accelerates atherogene-sis in an apoE-deficient mouse model. Female apoE-deficient mice were subjected to multiple low-doseintraperitoneal injections of STZ. As a control, sustained-release insulin implants were inserted in a subgroup ofSTZ-injected mice. Plasma glucose levels were checkedafter the last STZ injection (data not shown) and weresignificantly higher than controls at the time of death

FIG. 1. A: Northern blot analysis of steady-state mRNA levels of GRP78/BiP in HepG2, HASMC, and Thp-1 cells exposed to glucose, glucosamine,or mannitol for 4 h, as indicated. The relative amount of mRNA normalized to 18S RNA and averaged over three independent experiments isindicated under each lane. B: Immunoblot analysis of GRP78/BiP levels in HepG2 cells pretreated for 15 min with 0 or 20 �mol/l azaserine andthen cultured in the presence of glucose or glucosamine, as indicated above, for 4 h. C: HepG2 cells were treated with the O-GlcNAcase inhibitorPUGNAc or glucosamine for 8 h, as indicated. Blots were immunostained with antibodies against O-GlcNAc and GADD153/CHOP. The relativeamount of protein normalized to �-actin averaged over three independent experiments is indicated under each lane in B and C.

FIG. 2. HASMC and HepG2 cells were cultured in the presence of ERstress agents A23187 (10 �mol/l), tunicamycin (2 �g/ml), or glu-cosamine (5 mmol/l), as indicated. After 24 h, cells were fixed andstained with filipin. Intracellular filipin-cholesterol complexes werevisualized and quantified using ImageQuant software. *P < 0.05 rela-tive to untreated control.

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(Table 1). Triglyceride and total cholesterol levels in15-week-old hyperglycemic mice tended to be higher thanin control mice; however, the differences were not statis-tically significant. The large standard deviation in totalplasma cholesterol concentration in the hyperglycemicmice is most likely an early indication of the dyslipidemiathat is observed in 20-week-old mice. Lipid profiles werealso very similar in control and diabetic 15-week-old mice.Significant differences in total cholesterol and lipid pro-files were observed in 20-week-old diabetic mice (Table 1and Fig. 4). Specifically, VLDL and intermediate-densitylipoprotein/LDL levels were elevated in 20-week-old hyper-glycemic mice, a finding that is consistent with previousreports (32). At 15 weeks of age, the hyperglycemic micehad significantly advanced atherosclerosis relative to con-

trols (Fig. 4). Mice with the slow-release insulin implanthad significantly smaller atherosclerotic lesions.Elevated O-GlcNAc in tissues from STZ-induced hy-

perglycemic apoE-deficient mice. To estimate the rela-tive intracellular concentration of glucosamine, wemonitored the extent of O-GlcNAc modification of cellularproteins by immunostaining techniques, as previously de-scribed (28). Immunohistochemical and immunoblot anal-ysis of liver, aorta, skeletal muscle, and epidydimal fat pad,using an antibody directed against O-GlcNAc (CTD110.6),indicates increased levels of intracellular glucosamine inSTZ-injected hyperglycemic mice compared with controls(Fig. 5). Treatment of hyperglycemic mice with subcuta-neously inserted sustained-release insulin implants (0.1unit/day) appeared to reduce intracellular glucosaminelevels in liver and aorta but increase O-linked glycosyla-tion in the adipocytes of the fat pad. This is likely a result ofthe 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 anddiabetic apoE-deficient mice were examined to determinewhether there is a correlation between intracellular glu-cosamine levels, ER stress, and complications of diabetes,including hepatic steatosis and atherogenesis. Liver tissuefrom hyperglycemic mice showed lipid accumulation andincreased staining for ER stress markers in hepatocytes(Fig. 6A). This result is consistent with our findings thatHepG2 cells treated with glucosamine and other ERstress–inducing agents show significant cholesterol accu-mulation. The intensity of both the KDEL and Oil Red Ostaining appeared to be lower in the insulin-treated hyper-glycemic mice.

Atherosclerotic lesions in the diabetic mice stainedmore intensely for the ER stress markers phospho-PERKand KDEL, especially in intimal macrophages and macro-phage foam cells (Fig. 6B). A comparison of lesions from15- and 20-week-old mice suggests that ER stress levels areindependent of lesion size (data not shown). Insulin treat-ment appeared to decrease the intensity of staining for ERstress markers in the hyperglycemic mice, a finding con-sistent with reduced intracellular glucosamine levels ob-

FIG. 3. HepG2 cells were transfected with a reporter plasmid contain-ing a luciferase gene driven by promoters containing NF-�B or serumresponse factor (SRF)-binding elements. After 24 h, cells were treatedwith glucosamine (5 mmol/l) or tunicamycin (2 �g/ml) for an additional8 h. Cell extracts were prepared and luciferase activity quantified. Thedata represent the averages � SD of three experiments, each per-formed in triplicate. *P < 0.05 relative to untreated controls.

TABLE 1Female apoE-deficient mice subjected to multiple low-dose intraperitoneal injections of STZ and, as a control, sustained releaseinsulin implants inserted in a subgroup of STZ-injected mice

Control STZ STZ � insulin

Tissuesn 13 13 6Body mass (g) 19.4 � 0.9 17.6 � 1.5* 17.4 � 2.1*Liver mass (g) 0.83 � 0.11 0.96 � 0.11* 0.84 � 0.16†Fat pad mass (g) 0.16 � 0.01 0.068 � 0.022* ND

Plasman 18 18 10Glucose (mmol/l) 10.3 � 2.6 18.5 � 2.6* 16.7 � 5.6‡Triglycerides (mmol/l) 0.92 � 0.31 1.00 � 0.18† 1.45 � 0.80†Cholesterol (mmol/l)

15-week-old mice 9.4 � 1.1 12.0 � 6.4† 13.8 � 7.0†20-week-old mice 19.4 � 2.3 30.4 � 1.8* ND

Lesionn 18 18 10Lesion area (104 �m2) 5.4 � 2.2 10.0 � 3.4‡ 3.9 � 0.8†

Data are means � SE unless otherwise indicated. *P � 0.01 relative to corresponding control; †not significant; ‡P � 0.05 relative tocorresponding control. ND, no data.

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served in Fig. 5. Coimmunofluorescence using an antibodyagainst O-GlcNAc and anti–phospho-PERK indicates thatthere is a strong colocalization of markers of intracellu-lar glucosamine and ER stress in the atheroscleroticlesions of hyperglycemic apoE-deficient mice (Fig. 6C).This observation supports our overall hypothesis thatglucosamine-induced ER stress promotes acceleratedatherogenesis.

DISCUSSION

A great deal of research has gone into the investigation ofmolecular and cellular mechanisms by which hyperglyce-

mia may promote the development of atherosclerosis.Much of this has focused on the interaction of AGE withthe receptor RAGE, which has been shown to trigger theproduction of intracellular reactive oxidative species andinitiate inflammatory pathways. Evidence supporting ac-celerated atherosclerosis through AGE-induced inflamma-tion has been obtained in a variety of experimentalsystems (33,34). However, the role of AGE in atherogene-sis is confounded by the apparent inability of antioxidantsupplementation to improve cardiovascular risk in dia-betic patients, as demonstrated by several large well-controlled clinical trials (7–10). This paradox supports the

FIG. 4. A: Immunohistochemical staining of representative pancreatic islets from saline- and STZ-injected apoE-deficient mice. Pancreaticsections were stained with an antibody against glucagon. B: Representative aortic cross-sections from 15-week-old female apoE-deficient miceinjected with multiple low doses of saline or STZ as indicated. C: Quantification of lesion area in control mice, STZ-injected mice, andSTZ-injected mice with insulin implants. *P < 0.05 relative to control, n � 13, for each group. D: Lipid profiles of STZ-injected and controlapoE-deficient mice. Plots represent the average values from three mice per group.

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possibility that other AGE-independent mechanisms andpathways may play an important role in the atherogenicprocess. Here, we present evidence for a novel mechanismby which the accumulation of intracellular glucosamine,observed in hyperglycemic conditions, induces ER stressin cell types relevant to the development of atherogenesis.The cellular reaction to ER stress initiates a multifaceted,cell-specific response that can include the dysregulationof lipid metabolism and inflammation, ultimately result-ing in accelerated atherosclerosis and hepatic steatosis(18,23,24).

Under physiological conditions, only 1–3% of intracellu-lar glucose enters the hexosamine pathway; however, fluxincreases with glucose concentration (11,12). Increasedhexosamine pathway activity and corresponding elevatedglucosamine levels have been implicated in several diabe-tes-associated complications, including insulin resistance(12), pancreatic �-cell death (13), and atherosclerosis (14).

It is not clear how intracellular glucosamine promotescellular dysfunction, but most research has focused on theO-linked glycosylation of serine and threonine residues ofspecific proteins, including transcription factors, nuclearpore proteins, and signaling factors (28,35,36).

An additional intracellular effect of glucosamine thathas not been investigated in the context of diabetes andatherosclerosis is its ability to promote the accumulationof unfolded proteins in the ER, a condition defined as ERstress (17). In mammals, ER stress triggers the activationof the unfolded protein response, involving three distinctintegral ER membrane proteins, designated PERK, Ire1(inositol-requiring ER-to-nucleus signal kinase 1), andATF6 (activating transcription factor 6) (rev. in 37). To-gether, these proteins signal the general inhibition ofprotein expression and the specific induction of ER-resident chaperone expression, including GRP78/BiP (37).The ER stress response also induces expression of the

FIG. 5. A: Immunohistochemical staining of O-GlcNAc–modified proteins in tissue sections from control, STZ-injected, and STZ- plusinsulin-treated apoE-deficient mice. B: Immunoblot analysis of O-GlcNAc–modified proteins in lysates from HepG2 cells exposed to elevatedconcentrations of glucosamine (GlcN) and liver from control (C), STZ-injected (S), and insulin-treated hyperglycemic mice (SI). The nuclear poreprotein, p62, is indicated. The number under each lane represents the relative amount of p62-O-GlcNAc normalized to �-actin.

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transcription factor GADD153/CHOP, which is known toplay a role in ER stress-induced growth arrest and pro-grammed cell death (38). The balance of protective andproapoptotic signals triggered by ER dysfunction deter-mines the ultimate fate of a cell. We have demonstratedthat both glucose and glucosamine can promote ER stressin cultured hepatocytes, monocytes, and smooth musclecells. In support of our in vitro findings, ER stress (39),alterations in ER morphology (17), and disruptions in theER trafficking of specific proteins (40) have been previ-ously observed in cultured cells exposed to glucosamineand in hyperglycemic mice. Our data suggests that theability of glucosamine to induce ER stress is independentof increased O-linked glycosylation. This finding is signif-icant because it supports a role for hyperglycemia-associ-ated, glucosamine-induced ER stress that is distinct frompreviously identified effects of O-linked protein glycosyla-tion, which include insulin resistance (41,42).

In recent years ER stress has been shown to affect lipidmetabolism through the activation and dysregulation ofthe SREBPs (24,30,43). SREBPs regulate the expression ofenzymes required for cholesterol and fatty acid biosynthe-sis and uptake (44). ER stress–inducing agents have alsobeen shown to activate NF-�B, the transcription factorthat regulates expression of inflammatory proteins (31).Together, inflammation and lipid accumulation representthe predominant characteristics of atherosclerosis. Li et al.(45) have recently shown that the loading of culturedmouse peritoneal macrophages with free cholesterol cancause ER stress that subsequently plays an essential rolein the induction of inflammatory pathways. Together withour results, this finding suggests that ER stress and lipidaccumulation may work through mutually reinforcingpathways that ultimately give rise to the inflammatoryproperties of lipid-engorged macrophages.

We have previously shown that hyperhomocysteinemia,

FIG. 6. A: Liver sections from control, hyperglycemic, and hyperglycemic mice with insulin implants immunostained with antibodies against KDELor stained with Oil Red O as indicated. B: Cross-sections of aortic root immunostained with antibodies specific for the phosphorylated form ofPERK and KDEL. C: Colocalization of antibodies against O-GlcNAc (CTD110.6) and phospho-PERK in a lesion from a hyperglycemicapoE-deficient mouse.

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a recognized independent risk factor for atherosclerosis,can induce ER stress in HASMCs and hepatocytes. Homo-cysteine-induced ER stress is associated with the dysregu-lation of lipid metabolism through the unregulatedactivation of SREBPs (24) and the induction of proapop-totic pathways (46). Hyperhomocysteinemic mice developaccelerated atherosclerosis, with lesions that show indica-tions of elevated levels of ER stress markers, includingphospho-PERK and GRP78 (18). Thus, elevated diagnosticmarkers of ER stress are a common characteristic of twodistinctly different models of accelerated atherosclerosisinvolving diabetes and hyperhomocysteinemia. This obser-vation suggests that the ER stress pathway may representa common or unifying mechanism of atherogenesis. Insupport of this hypothesis, obesity, an independent riskfactor for atherosclerosis, has recently been shown topromote ER stress in cell culture and mouse modelsystems (47). It is important to note that although hyper-glycemia and hyperhomocysteinemia accelerate athero-genesis in apoE-deficient mice, a strain predisposed tolesion development, these conditions do not cause athero-sclerosis in wild-type mice (18,19). This observation sug-gests that factors in addition to ER stress also contributeand are required for lesion development.

The effects of hyperglycemia-associated ER dysfunctionare evident in both the liver and the vascular cells of theaortic wall. Therefore, we cannot determine, at this time,the relative importance of a local dysregulation of lipidmetabolism in vascular cells and a distal hepatic disrup-tion of lipid metabolism. In 15-week-old diabetic mice,total plasma cholesterol and VLDL, intermediate-densitylipoprotein, and LDL cholesterol levels, in particular, arenot significantly different from age-matched nondiabeticapoE-deficient mice. This finding suggests that plasmalipid levels do not play a major role in early stages ofaccelerated lesion development in this model. The poten-tial for ER stress to act locally in cells of the artery wall topromote atherosclerosis is also supported by our observa-tion that cultured human monocytes and aortic smoothmuscle cells respond to glucose/glucosamine-induced ERstress by accumulating free cholesterol. At this time we donot know whether the accumulation of lipids in vascularcells is a result of increased biosynthesis and/or increaseduptake. Our previous work suggesting that the ER stressdysregulates SREBPs, transcription factors that controlthe expression of lipid biosynthetic enzymes as well asLDL receptors, would support a role for both pathways(24).

Identification of a role for ER stress in the developmentand progression of atherosclerosis is important to ourunderstanding of the molecular mechanisms that linkhyperglycemia to vascular disease. Studies are now under-way to determine the vascular effects of manipulating ofER stress signaling pathways in hyperglycemic mice.

ACKNOWLEDGMENTS

Funding from the Canadian Institutes of Health Research(MOP-62910) and the Heart and Stroke Foundation ofOntario (NA5556) supported this work. G.H.W is sup-ported by a Heart and Stroke Foundation of Canada newinvestigators grant.

We thank Dr. Ji Zhou and Colin Halford for experttechnical advice and assistance.

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