ceramide mediates inhibition of the akt/enos pathway by high levels of glucose in human vascular...

DOI 10.1515/jpem-2012-0144 J Pediatr Endocr Met 2013; 26(1-2): 31–38

Aimin Wang , Chun Li , Jie Liao , Min Dong , Zhiming Xiao and Minxiang Lei *

Ceramide mediates inhibition of the Akt/eNOS pathway by high levels of glucose in human vascular endothelial cells

Abstract Objective: To investigate how ceramide mediates the

effects of high-glucose-induced inhibition of the Akt/

endothelial nitric oxide synthase (eNOS) signalling

pathway in human vascular endothelial cells (HUVECs).

Materials and methods: NO levels were determined by

ELISA. Endogenous ceramide levels were determined

using a liquid chromatography-mass spectrometry assay.

Akt and eNOS protein expressions were determined by

Western blotting.

Results: High-glucose levels induce ceramide accumula-

tion in a dose- and time-dependent manner (p < 0.05). We

also show that exposure of HUVECs to high-glucose con-

ditions inhibits the insulin-mediated activation of Akt/

eNOS signalling and the subsequent NO generation in a

dose-dependent manner (p < 0.05). Preventing de novo

ceramide synthesis attenuated the antagonistic effects of

high-glucose levels on the Akt/eNOS signalling pathway

(p < 0.05); conversely, inducing ceramide build-up aug-

mented the inhibitory effects of high-glucose levels on the

Akt/eNOS signalling pathway (p < 0.05).

Conclusion: Ceramide is both necessary and sufficient

for mediating the inhibition of the Akt/eNOS signalling

pathway by high-glucose levels in endothelial cells.

Keywords: Akt/endothelial nitric oxide synthase (eNOS);

ceramide; glucose; nitric oxide (NO); type 2 diabetes mellitus.

*Corresponding author: Minxiang Lei, Department of Endocrinology,

Xiangya Hospital, Central South University, Xiangya Road, 87 # ,

Changsha, Hunan 410008, China, Phone: + 86-731-84327089,

Fax: + 86-731-84327089, E-mail: [email protected]

Aimin Wang: Department of Endocrinology , Xiangya Hospital,

Central South University, Changsha, Hunan, China

Chun Li: Department of Endocrinology , Xiangya Hospital, Central

South University, Changsha, Hunan, China

Jie Liao: Department of Endocrinology , Xiangya Hospital, Central

South University, Changsha, Hunan, China

Min Dong: School of Pharmaceutical Sciences , Guangxi Medical

University, Nanning, Guangxi, China

Zhiming Xiao: The Third Hospital of Xiangya , Central South

University, Changsha, China

Introduction Type 2 diabetes mellitus is characterised by chronic hyper-

glycaemia and inadequate β -cell responses to progressive

insulin resistance (1, 2) . Chronic hyperglycaemia is the

proximate cause of many diabetic complications, includ-

ing retinopathy, kidney failure, neuropathies, and vascu-

lar disease (3, 4) . Chronic hyperglycaemia also has toxic

effects on the vascular endothelium, another target of dia-

betic complications (5) . Oxidative stress induced by high-

glucose levels is a common pathophysiological pathway

found in diabetic complications, especially with respect

to vascular disease.

The vascular endothelium is an insulin-sensitive

tissue; in vascular endothelial cells, the phosphati-

dylinositol 3 kinase (PI3K) pathway activates the serine/

threonine protein kinase Akt, which phosphorylates

endothelial nitric oxide synthase (eNOS) leading to

increased nitric oxide (NO) production (6, 7) . Changes

in NO levels are the earliest known characteristic of

dysfunctional vascular endothelial cells (8, 9) . There-

fore, downregulation of insulin signalling can lead to

metabolic and cardiovascular dysfunction. Ceramide, a

metabolic byproduct of the signalling molecule sphin-

gomyelin, has been widely implicated in the regula-

tion of intracellular signalling pathways, and it plays

important roles in cell growth, proliferation, motility,

adhesion, differentiation, senescence, and apoptosis

(10 – 13) . As a second-messenger lipid, ceramide is sig-

nificantly associated with insulin resistance induced

by tumour necrosis factor- α (TNF- α ) and free fatty acid

(FFA) (14, 15) . Ceramide also blocks the insulin recep-

tor substrate-1 (IRS-1)/PI3K/eNOS signalling pathway at

several different stages. Our previous studies have dem-

onstrated that FFA can induce ceramide accumulation

in human umbilical vein endothelial cells (HUVECs),

leading to reductions in eNOS phosphorylation and

vascular endothelial dysfunction (16) . Dandan et al.

demonstrated that high-glucose levels could induce

apoptosis in vascular endothelial cells and increase

intracellular ceramide accumulation (17) . Chun et al.

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32 Wang et al.: Ceramide and Akt/eNOS signalling pathway

found that inhibition of de novo ceramide synthesis

improved endothelium-dependent vasodilation (EDVD)

of the arteries of streptozotocin-induced diabetic rats

(18) . Therefore, ceramide accumulation is an important

mediator associated with vascular endothelial dysfunc-

tion. However, it remains unclear whether ceramide

accumulation causes high-glucose-induced endothelial

dysfunction through targeting of the insulin-induced

Akt/eNOS signalling pathway.

Ceramide biosynthesis requires the coordinated

action of two enzymes: serine palmitoyltransferase and

ceramide synthase (19) . Serine palmitoyltransferase

catalyses the initial step, which involves the condensa-

tion of serine and palmitoyl-CoA to form 3-keto-sphin-

ganine. Studies have shown that high-glucose-induced

mechanical dysfunction can be significantly attenu-

ated by both the membrane-permeant ceramide analog

C2-ceramide or the ceramide glucosyltransferase inhibi-

tor D, L-threo-1-pheny-2-decanoylamino-3-morpholino-

1-propanol (PDMP). We hypothesised that ceramide

might be the principal effector molecule that mediates

high-glucose-induced Akt/eNOS inhibition. Therefore,

we first determined whether myriocin and desipramine

(DES) – inhibitors of de novo ceramide synthesis – could

prevent high-glucose-induced inhibition of the Akt/PKB

signalling pathway. Next, we determined whether PDMP

and N-oleoylethanolamine (NOE) – specific inhibitors

of ceramide glucosyltransferase – could also influence

high-glucose-induced inhibition of the Akt/PKB signal-

ling pathway.

Based on the experiments described above, we inves-

tigated how ceramide affects the high-glucose-induced

inhibition of the Akt/eNOS signalling pathway in cultured

HUVECs. Our study suggests that ceramide is an impor-

tant mediator molecule that plays a crucial role in high-

glucose-induced inhibition of the Akt/eNOS signalling

pathway.

Materials and methods

Cell culture HUVECs were cultured in Dulbecco ’ s modified eagle media

(DMEM) supplemented with 10 % foetal calf serum (FCS). The cells

were maintained at 37 ° C under a humidified atmosphere of 5 % CO 2

and 95 % air in a Forma Scientific incubator (Biosciences, Dublin,

Ireland). Differentiated HUVECs were seeded into six-well plates

at 5 × 10 5 cells per well and allowed to adhere overnight. When

cultures reached 90 % confluence, the supernatants were harvest-

ed and/or cell lysates were prepared for assaying as described

below.

High-glucose treatment and insulin stimulation First, HUVECs were exposed to 5 mmol/L D-glucose and 30 mmol/L

D-glucose (high glucose) medium for 4, 8, 12, 16, or 24 h to observe

ceramide accumulation levels. Next, HUVECs were incubated with

various concentrations of D-glucose (5, 10, 20, or 30 mmol/L) for 16 h

to observe ceramide accumulation levels and NO production. Finally,

HUVECs were stimulated with 50 nmol/L insulin for 30 min to observe

changes in NO levels. The groups were as follows: (i) HUVECs treated

with 25 mmol/L L-glucose and 5, 10, 20, or 30 mmol/L D- glucose;

(ii) to observe the changes of NO levels, HUVECs were stimulated with

50 nmol/L insulin for 30 min in media supplemented with 25 mmol/L

L-glucose and 5, 10, 20, or 30 mmol/L D-glucose; (iii) select samples

were treated with 10 μ mol/L myriocin or 2 μ mol/L DES prior to the ad-

dition of high levels (30 mmol/L) of glucose; and (iv) select samples

were treated with or without high levels of glucose in the presence of

the ceramidase inhibitor (NOE) (250 μ mol/L) or the glucosylceramide

synthase inhibitor (PDMP) (50 μ mol/L), which were added prior to

glucose addition.

Ceramide assays Confl uent HUVEC cultures were incubated in 175 cm 2 tissue culture

fl asks at 37 ° C for 30 min in 20 mL of serum-free DMEM, followed by

rinsing with the same medium. Next, the cells were detached with

0.05 % trypsin/0.53 mmol/L EDTA (Gibco-BRL) and pelleted by cen-

trifugation. The pellets were washed 3 times with 10 mL cold phos-

phate buff ered saline (PBS), followed by another centrifugation step.

Approximately 5 × 10 6 washed cells were resuspended in 220 μ L cold

0.25 mmol/L sucrose in PBS, transferred into microfuge tubes, and

disrupted by sonication. The cell lysates were then centrifuged at

800 × g for 10 min, and the supernatants containing the cytosolic

fraction – made up of the cytoplasm and organelles – were with-

drawn. The pellets containing the membrane fractions were then

washed 3 times with 1 mL cold PBS, centrifuged, and suspended in

220 μ L PBS. Ceramide levels in each of the above fractions were de-

termined using the methods described by Zhou et al. (20) . Deriva-

tised ceramide was separated from byproducts using 3 % 2-propanol

in n-hexane as the mobile phase. The fl ow rate was 2.0 mL/min, and

the eluted compounds were monitored using a fl uorescence spectro-

photometer with an excitation wavelength of 230 nm and emission

wavelength of 352 nm.

Determination of NO concentrations Although NO is unstable, its end products – nitrite and nitrate –

are stabile. Therefore, in order to index NO generation, we deter-

mined the sum of the arterial concentrations of nitrite and nitrate.

The culture media were harvested and stored at – 80 ° C until used

in the assays. Nitrite and nitrate levels were measured as described

previously (21) . Briefl y, nitrate was converted to nitrite with nitrate

reductase, and total nitrite was reacted to the Griess reagent; the

coloured product could then be quantifi ed by its absorbance at 540

nm using a spectrophotometer. Each experiment was repeated in

triplicate.



Wang et al.: Ceramide and Akt/eNOS signalling pathway 33

Western blot analyses To obtain total protein, cell extracts were prepared in lysis buff er con-

taining 50 mmol/L Tris HCl (pH 8.0), 150 mmol/L NaCl, 0.1 % SDS, 0.1

mmol/L phenylmethylsulphonyl fl uoride (PMSF), 10 lg/mL aprotinin,

1 % Nonidet (N)P-40, and 0.5 % sodium deoxycholate. The lysates

were cleared by centrifugation at 12,000 × g for 10 min at 4 ° C. Total

protein concentrations were measured using the Bradford method

(22) . The proteins were separated on 10 % SDS-PAGE gels, transferred

to polyvinylidene difl uoride membranes, and then probed with pri-

mary antibodies against Akt, eNOS, or GADPH; all primary antibod-

ies were polyclonal rabbit antibodies from Santa Cruz Biotechnology.

Evaluations were performed with the Fluor Chem. 8900 soft ware

system.

Statistical analyses All statistical analyses were carried out using SPSS soft ware (version

13.0 for Windows; SPSS, Chicago, IL, USA). Statistical signifi cance of

the mean values was evaluated using a one-way analysis of variance

(ANOVA) test for measurements and an LSD-t-test for comparisons

between datasets. All data are presented as means ± standard deviation

(SD). A p-value < 0.05 was considered to be statistically signifi cant.

Results

Time- and dose-dependent eff ects of D- glucose induction on ceramide accumulation

First, HUVECs were divided into control (5 mmol/L

D- glucose) and high-glucose-treated (30 mmol/L

D- glucose) groups, which were treated with medium for

4, 8, 12, 16, or 24 h. We found that ceramide levels were

significantly higher in the high-glucose-treated group

compared with the control group and that ceramide

accumulation peaked at 16 h (Figure 1 A). Next, HUVECs

were divided into five groups and treated with one of

the following: 5 (control), 10, 20, or 30 (high) mmol/L

D- glucose or 25 mmol/L L-glucose for 16 h. We found sig-

nificant increases in ceramide accumulation in HUVECs

treated with 10, 20, and 30 (high) mmol/L D-glucose for

16 h compared with the control group: ceramide accumu-

lation levels reached a maximum under the high-glucose

conditions (p < 0.001). However, we did not observe a sig-

nificant difference in ceramide accumulation between

cells treated with 25 mmol/L L-glucose and the control

group (p > 0.05) (Figure 1B). Our results demonstrate that

glucose induces ceramide accumulation in HUVECs in

both a time- and dose-dependent manner.

High-glucose levels inhibit activation of insulin-signalling components and basal insulin-stimulated eNOS activation

The Akt/eNOS signalling pathway in the vascular

endothelium involves Akt-mediated phosphorylation of

Ser1177 on eNOS, which results in increased eNOS activ-

ity and NO production. Because insulin activates eNOS

through Akt, we investigated whether high-glucose levels

have an effect on basal and insulin-stimulated Akt and

eNOS phosphorylation levels. To measure Akt and eNOS

phosphorylation, we used antibodies that detected total

Akt and eNOS protein levels as well as antibodies specific

100A B5 mM D-glu30 mM D-glu

#*#*#*

**

**

*

80

60

Cer

amid

e, n

g/10

6 cel

ls

40

20

Time, h

0

100

80

60

Cer

amid

e, n

g/10

6 cel

ls

40

20

0

4 h 8 h 12 h

16 h

24 h

5 mM D

-glu

10 m

M D-gl

u

20 m

M D-gl

u

30 m

M D-gl

u

25 m

M L-glu

Figure 1 Time- and dose-dependent effects of D-glucose (D-glu) induction on ceramide accumulation. (A) A comparison of ceramide

accumulation in HUVECs exposed to 5 or 30 mM (high glucose) D-glu at 4, 8, 12, 16, or 24 h. (B) A comparison of ceramide accumulation

in HUVECs exposed to 5, 10, 20, or 30 mM D-glu or 25 mM L-glu for 16 h. Data shown are the mean ± SD values from six measurements.

Asterisks (*) denote significant differences in ceramide accumulation with respect to different concentrations of D-glu (p < 0.05). Number

signs ( # ) denote significant differences in ceramide accumulation with respect to different times (p < 0.05).




to the Ser473- and Ser1177-phosphorylated forms of Akt

and eNOS. Our results demonstrate that high-glucose

levels can markedly inhibit insulin-stimulated Akt and

eNOS phosphorylation at Ser473 and Ser1177 in HUVECs

in a dose-dependent manner compared with cells not

treated with high-glucose levels (Figure 2 A). Impor-

tantly, we did not observe changes in the total levels of

Akt and eNOS protein expression under these conditions.

Our data suggest that high-glucose levels may block the

insulin-signalling pathway at both Akt activation and

eNOS phosphorylation steps. Next, we assessed changes

in NO production in HUVECs that were either pretreated or

treated with high levels of glucose, and our results show

that there were significant decreases in NO production

both with and without insulin treatment (Figure 2B,C).

These results suggest that high levels of glucose are able

to inhibit eNOS activity. Therefore, high-glucose levels can

inhibit both basal and insulin-stimulated types of eNOS

phosphorylation; this would lead to inactivation of eNOS

in endothelial cells, ultimately resulting in decreased NO

production.

High levels of glucose induce ceramide accumulation

We have demonstrated that high levels of glucose can

induce ceramide accumulation (Figure 1) in a dose- and

time-dependent manner. To observe the changes in intra-

cellular ceramide levels in these experiments, we utilised

several enzyme inhibitors to block ceramide synthesis.

Pretreating HUVECs with myriocin – a fungal toxin that

inhibits serine palmitoyltransferase – completely pre-

vented high-glucose-induced ceramide accumulation

(Figure 3 A). DES has been widely used in the literature

as a specific acid sphingomyelinase inhibitor (23) . Some

studies have demonstrated that DES produces a dose- and

time-dependent downregulation of acid ceramidase (24) .

In our studies, we found that DES abolished the high-

glucose-mediated effects on ceramide. Our results suggest

that high levels of glucose induce ceramide accumulation

over basal levels (p < 0.01) and that this process is medi-

ated by the actions of the two enzymes responsible for

ceramide biosynthesis (p < 0.05) (Figure 3A).

40

30

20

No,

μM

/L

10

5 5 10 20 30

p-Akt

A B

C

t-Akt

p-eNOS

t-eNOS

NADPH

D-glu, mM

Insulin- + + + +

0

40

30

20

*

* * *

**

No,

μM

/L

10

0

5 mM D

-glu

5 mM D

-glu+

Ins

10 m

M D-gl

u+Ins

20 m

M D-gl

u+Ins

30 m

M D-gl

u+Ins

25 m

M L-glu

+Ins

10 m

M D-gl

u

20 m

M D-gl

u

30 m

M D-gl

u

25 m

M L-glu

Figure 2 Effects of D-glucose (D-glu) on insulin-stimulated Akt and eNOS phosphorylation. HUVECs were incubated in the presence of

5, 10, 20, or 30 mM D-glu or 25 mM L-glu medium for 16 h before and after stimulation with insulin at 50 nM for 30 min. Cell lysates were

resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. (A) A Western blot

analysis with antibodies against phosphorylated Akt and total Akt, and against phosphorylated eNOS and total eNOS. (B) A comparison of

the changes in NO levels seen in HUVECs exposed to 5, 10, 20, or 30 mM D-glu at 16 h. (C) A comparison of the changes in NO levels seen in

HUVECs stimulated with 50 nM insulin for 30 min. Data shown are mean ± SD values from six measurements. Asterisks (*) denote significant

changes in NO levels with respect to different concentrations of D-glu (p < 0.05).




Ceramide mediates high-glucose-induced inhibition of the Akt/eNOS pathway

We found that myriocin completely inhibited ceramide

accumulation (p < 0.05) (Figure 3A) and increased Akt and

eNOS phosphorylation under high-glucose conditions

(p < 0.05) (Figure 3C). Similarly, another inhibitor, DES,

could also prevent the effects of high levels of glucose on

ceramide accumulation (p < 0.05) (Figure 3A) and increase

Akt and eNOS phosphorylation (p < 0.05) (Figure 3D). At

the same time, NO production was significantly increased

compared with the high-glucose group (p < 0.05) (Figure

3B). Therefore, both myriocin and DES are able to protect

Akt and eNOS phosphorylation from the inhibitory effects

of high glucose.

Next, we wanted to determine whether the gluco-

sylceramide synthase inhibitor PDMP or the ceramidase

inhibitor NOE could prevent inhibitory effects of high levels

of glucose on Akt and eNOS phosphorylation. Therefore,

we treated cells with either PDMP or NOE, which increase

endogenous ceramide levels by blocking ceramide degra-

dation. Treating HUVECs with either compound increased

cellular ceramide levels in a similar manner to high-glu-

cose medium (Figure 4 A,B); furthermore, NOE markedly

inhibited insulin-stimulated Akt and eNOS phosphoryla-

tion (Figure 4C) but did not affect the total levels of Akt

or eNOS protein expression. In other words, increasing

endogenous ceramide levels using alternative methods

can recapitulate the inhibitory effects of high levels of

glucose on the insulin-signalling pathway. Our data also

100A

C D

B

# ##

#

**

*

80

40

30

20

No,

μM

/L

10

p-Akt

t-Akt

p-eNOS

t-eNOS

NADPH

Insulin- + + +

High glucose- - + +

Myriocin- - - +

p-Akt

t-Akt

p-eNOS

t-eNOS

NADPH

Insulin- + + +

High glucose- - + +

DES- - - +

Basal Ins

30 m

M D-gl

u+Ins

30 m

M D-gl

u+Ins

+Myri

ocin

30 m

M D-gl

u+Ins

+DES

0

60

40

Cer

amid

e, n

g/10

6 cel

ls

20

0

Contro

l

25 m

M L-glu

30 m

M D-gl

u

30 m

M D-gl

u+Myri

ocin

30 m

M D-gl

u+DES

Figure 3 Effects of myriocin or desipramine (DES) on high-glucose-mediated inhibition of insulin-stimulated Akt and eNOS phosphoryla-

tion, ceramide accumulation, and NO production. HUVECs were incubated under high-glucose conditions for 16 h before stimulation with

50 nM insulin for 30 min. Select samples were treated with 10 μ M myriocin or 2 μ M DES prior to treatment with (or without) high levels of

glucose. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies against

phosphorylated Akt or eNOS. Data shown are representative of at least six independent experiments. (A) Measurements of ceramide

accumulation in the culture supernatants of HUVECs that were pretreated as indicated. (B) Measurements of NO concentration in culture

supernatants of HUVECs that were pretreated as indicated. (C, D) Western blot analyses with antibodies against phosphorylated Akt and

total Akt, and against phosphorylated eNOS and total eNOS. Data shown are mean ± SD values from six measurements. Asterisks (*) denote

that the values obtained were significantly different from basal levels (p < 0.05). Number signs ( # ) denote that the values obtained were

significantly different from the high-glucose-treated group.




demonstrate that PDMP and NOE can augment the effects

of high-glucose-induced NO generation by blocking cera-

mide glucosylation or deacylation.

Discussion The study reported here demonstrates that ceramide plays

an important role in high-glucose-induced inhibition of

the Akt/eNOS signalling pathway in vascular endothelial

cells. Our findings include the following: (i) high levels of

glucose induce ceramide accumulation; (ii) high levels of

glucose inhibit Akt and eNOS activity through the Akt/

eNOS signalling pathway in cultured HUVECs, leading to

significant decreases in NO generation; (iii) the inhibitory

effects of high levels of glucose on Akt/eNOS signalling

pathway activation can be reversed by inhibiting de novo

ceramide synthesis; and (iv) blocking ceramide metabo-

lism both increases ceramide accumulation and inhibits

the Akt/eNOS pathway. Our findings suggest that cera-

mide is a key effector molecule that links high levels of

glucose to endothelial cell dysfunction through the Akt/

eNOS signalling pathway.

There exist many potential mechanisms whereby high

levels of glucose might induce endothelial cell damage.

High levels of glucose can cause chronic oxidative stress,

which in turn leads to the apoptosis of vascular endothelial

cells. The Akt/eNOS pathway involves a series of phospho-

rylation reactions dependent on insulin signalling (25, 26) .

100A

C

# #

*80

p-Akt

t-Akt

p-eNOS

t-eNOS

NADPH

- + + + Insulin

- - - 30 D-glu

NOE- - + -

+

30

+

60

40

Cer

amid

e, n

g/10

6 cel

ls

20

0

Contro

l

25 m

M L-glu

30 m

M D-gl

u

30 m

M D-gl

u+NOE

30 m

M D-gl

u+PDMP

B

* * *

40

30

20

No,

μM

/L

10

Basal Ins

30 m

M D-gl

u+Ins

30 m

M D-gl

u+Ins

+NOE

30 m

M D-gl

u+Ins

+PDMP

0

Figure 4 Effects of inhibitors on high-glucose-mediated inhibition of insulin-signalling component phosphorylation, ceramide accumula-

tion, or NO production. HUVECs were incubated in 30 mM D-glu (high glucose) conditions for 16 h before stimulation with 100 nM insulin

for 10 min. Select samples were treated either with or without high glucose in the presence of NOE (250 μ M) or PDMP (50 mM). Cell lysates

were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies against phosphorylated Akt or

eNOS. Data shown are representative of at least four independent experiments. (A) Western blot analyses with antibodies against phos-

phorylated Akt and total Akt. (B) Western blot analyses with antibodies against phosphorylated eNOS and total eNOS. (C) Measurements of

ceramide accumulation in culture supernatants of HUVECs that were pretreated as indicated. Measurements of NO concentrations in culture

supernatants of HUVECs that were pretreated as indicated. Data shown are mean ± SD values from six measurements. Asterisks (*) denote

that the values obtained were significantly different from the high-glucose-treated group (p < 0.05). Number signs ( # ) denote that the values

obtained were significantly different from the high-glucose-treated group.




After insulin binds to the insulin receptor (IR) on the cell

surface, the IR becomes activated and phosphorylates

IRS-1 by PTK inside the cell, which then phosphorylates

IRS-1 and PI3K, which in turn phosphorylate and activate

Akt (27, 28) . Because phosphorylated Akt is the kinase

that directly phosphorylates eNOS, Akt phosphorylation

levels may be an indicator of eNOS activity; therefore, we

investigated the effect of high levels of glucose on basal

and insulin-stimulated Akt activation by monitoring Akt

phosphorylation at Ser473. We observed that high levels

of glucose inhibited Akt phosphorylation in a dose-

dependent manner and also inhibited insulin-stimulated

eNOS phosphorylation/activation. We also showed that

high levels of glucose could inhibit eNOS activity through

the Akt/eNOS pathway; this resulted in a significant

decrease in NO generation, which could promote the

onset of endothelial dysfunction. High levels of glucose

inhibited eNOS phosphorylation at Ser1177 and activation

in a dose-dependent manner and also inhibited insulin-

stimulated eNOS phosphorylation. Because NO is a key

regulator of endothelial function, eNOS inhibition by high

levels of glucose could provide a mechanism to explain

endothelial dysfunction in diabetic vascular compliance.

Our results suggest that high levels of glucose are likely

to be a major risk factor for diabetic vascular compliance,

consistent with previous reports.

Ceramide is capable of inhibiting both Akt and Akt-

mediated eNOS phosphorylation. Our findings demon-

strate that inhibiting de novo ceramide synthesis can

prevent high-glucose-induced ceramide accumulation as

well as its antagonistic effects on the Akt/eNOS pathway,

resulting in eNOS activation and NO generation. We also

found that blocking ceramide metabolism while simul-

taneously adding high levels of glucose augmented both

ceramide accumulation and the phosphorylation of Akt

and eNOS.

Several convincing studies have demonstrated

that high levels of glucose cause increases in ceramide

concentration and endothelial cell apoptosis (17, 18) . In

this study, we employed several inhibitors to block spe-

cific enzymes involved in ceramide metabolism. First, the

fungal toxins myriocin and DES inhibit separate enzymes

required for de novo ceramide synthesis; both toxins

were able to protect HUVECs from ceramide accumula-

tion induced by high glucose. Furthermore, both of the

ceramide glucosyltransferase inhibitors PDMP and NOE,

which inhibit glucosylceramide synthase and ceramidase,

respectively, were able to induce ceramide accumulation.

Our data indicate that high levels of glucose can induce

ceramide accumulation and that ceramide is an important

mediator molecule associated with inhibition of the high-

glucose-induced Akt/eNOS pathway.

In summary, we demonstrate that high levels of

glucose induce ceramide accumulation, and we report

the novel finding that ceramide plays an important role

with respect to the inhibition of the Akt/eNOS pathway

by high levels of glucose. Further efforts will be neces-

sary to explore the precise mechanisms behind diabetic

vascular compliance and high-glucose-induced vascu-

lar endothelial cell dysfunction as mediated by the Akt/

eNOS signalling pathway on both the cellular and organ-

ismal levels.

Acknowledgements: We would like to thank all of the par-

ticipants in this study. This work was supported by the

National Natural Science Foundation of China (30871190)

and the Hunan Provincial Finance Department and Edu-

cation Steering Committee under grant no. (2009).

Conflict of interest statement The authors declare no conflicts of interest.

Received May 4, 2012; accepted September 21, 2012; previously

published online November 9, 2012

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