Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 2007;116:1585-1595
Published online before print September 17, 2007, doi: 10.1161/CIRCULATIONAHA.107.716498
CLINICAL PERSPECTIVE
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
116/14/1585    most recent
CIRCULATIONAHA.107.716498v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Song, P.
Right arrow Articles by Zou, M.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, P.
Right arrow Articles by Zou, M.-H.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
*Genetics Home Reference
Hazardous Substances DB
*L-SERINE
Related Collections
Right arrow Type 1 diabetes
Right arrow Oxidant stress
Right arrow Type 2 diabetes
Right arrow Endothelium/vascular type/nitric oxide

(Circulation. 2007;116:1585-1595.)
© 2007 American Heart Association, Inc.

Molecular Cardiology

Reactive Nitrogen Species Induced by Hyperglycemia Suppresses Akt Signaling and Triggers Apoptosis by Upregulating Phosphatase PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) in an LKB1-Dependent Manner

Ping Song, PhD; Yong Wu, MD, PhD; Jian Xu, PhD; Zhonglin Xie, MD, PhD; Yunzhou Dong, PhD; Miao Zhang, MD, PhD; Ming-Hui Zou, MD, PhD

From the Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City.

Correspondence to Ming-Hui Zou, MD, PhD, BSEB 325, Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, OK 73104. E-mail ming-hui-zou{at}ouhsc.edu

Received May 22, 2007; accepted July 23, 2007.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background— Oxidative stress plays a causal role in vascular injury in diabetes mellitus, but the mechanisms and targets remain poorly understood.

Methods and Results— Exposure of cultured human umbilical vein endothelial cells to either peroxynitrite (ONOO–) or high glucose significantly inhibited both basal and insulin-stimulated Akt phosphorylation at Ser473 and Akt activity in parallel with increased apoptosis, phosphorylation, and activity of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Furthermore, protein kinase B/Akt inhibition induced by ONOO– or high glucose and apoptosis triggered by high glucose could be abolished by transfection of PTEN-specific small interfering RNA, suggesting that PTEN mediated the Akt inhibition by ONOO–. In addition, exposure of human umbilical vein endothelial cells to ONOO– or high glucose remarkably increased Ser428 phosphorylation of LKB1, a tumor suppressor. Interestingly, the ONOO–-enhanced PTEN phosphorylation and Akt inhibition can be blocked by LKB1-specific small interfering RNA. Consistently, LKB1 phosphorylated PTEN at Ser380/Thr382/383 in vitro, suggesting that LKB1 might act as an upstream kinase for PTEN. Compared with nondiabetic mice, the levels of PTEN, LKB1-Ser428 phosphorylation, and 3-nitrotyrosine (a biomarker of ONOO–) were significantly increased in the aortas of streptozotocin-induced diabetic mice, which was in parallel with a reduction in Akt-Ser473 phosphorylation and an increase in apoptosis. Furthermore, administration of PTEN-specific small interfering RNA suppressed diabetes-enhanced apoptosis and Akt inhibition. Finally, treatment with Tempol, a superoxide dismutase mimetic, and insulin, both of which reduced the ONOO– formation, markedly reduced diabetes-enhanced LKB1-Ser428 phosphorylation, PTEN, and apoptosis in the endothelium of mouse aortas.

Conclusion— We conclude that hyperglycemia triggers apoptosis by inhibiting Akt signaling via ONOO–-mediated LKB1-dependent PTEN activation.


Key Words: apoptosis • endothelium • endothelium-derived factors • hyperglycemia • peroxynitrite


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The endothelium is thought to play a critical role in maintaining vascular homeostasis. Endothelial injury is well established in diabetes mellitus, as evidenced by increased levels of von Willebrand factor, abnormal overexpression of endothelial adhesion molecules, impaired endothelium-dependent relaxation, and accelerated apoptosis.1 Endothelial dysfunction also is detectable in healthy first-degree relatives of subjects with type 2 diabetes mellitus who are at increased risk of developing diabetes. The Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) have demonstrated the efficacy of intensive glucose control in reducing the risk of microvascular complications of diabetes such as retinopathy, neuropathy, and nephropathy.2 Recent findings from the long-term follow-up of participants in the DCCT also have demonstrated a decreased incidence of atherosclerosis in study participants who intensively controlled blood glucose, suggesting that hyperglycemia is important in the development of macrovascular diseases in diabetes mellitus.3 Mechanisms that may underlie hyperglycemia-induced vascular dysfunction include increased polyol pathway flux, altered cellular redox state, increased formation of diacylglycerol and the subsequent activation of specific PKC isoforms, and accelerated nonenzymatic formation of advanced glycation end products. Recently, Du and coworkers4 proposed overproduction of mitochondrial superoxide anions (O2•–) as the unifying mechanism for hyperglycemia-induced vascular injury. However, the molecular mechanisms tying hyperglycemia to vascular injury remain poorly understood.

Clinical Perspective p 1595

Activation of phosphoinositide-3'-kinase (PI3K) generates lipid products, including PI (3,4,5)P3, initiating a cascade of serine kinase activation whereby phosphoinositide-dependent kinase-1 (PDK-1) is phosphorylated, leading to the phosphorylation and activation of Akt, other serine kinases, and their downstream substrates. The PI3K–PDK-1–Akt axis plays essential roles in cell biology, including cell growth and cell death. In addition, this pathway ultimately culminates in the pleiotropic biological actions of insulin in vascular function. In addition to its known effects on metabolism, insulin, via the PI3K-Akt axis, increases nitric oxide (NO) release by phosphorylating serine (Ser) 1177 of endothelial NO synthase.5

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN), originally identified as a tumor suppressor gene mutated in a large percentage of human cancers,6 is considered to be a key negative regulator of the PI3K/Akt pathway.7,8 Previous studies have demonstrated that deletion of PTEN in muscle protects mice from insulin resistance and diabetes mellitus caused by high-fat feeding.9 Other studies provide evidence that increased PTEN might contribute to impaired NO release in endothelial cells exposed to either free fatty acids10 or resistin,11 although the mechanism by which diabetes mellitus activates PTEN remains unknown.

Peroxynitrite (ONOO–), a highly reactive oxidant formed by the diffusion-controlled reaction of O2•– and NO, is formed during sepsis, inflammation, diabetes mellitus, ischemia-reperfusion, and atherosclerosis and contributes to all of these pathophysiological processes.12–14 Hyperglycemia induces O2•– and ONOO– overproduction,4,15,16 and our laboratory has recently demonstrated that ONOO– dose dependently inhibits Akt activity.17 However, the underlying mechanism is not understood. Because PTEN has a critical role in antagonizing PI3K pathways, we reasoned that ONOO– generated by hyperglycemia blocks PI3K/Akt activation by upregulating PTEN. In the present study, we demonstrate that ONOO– significantly increased the phosphorylation of LKB1, a tumor suppressor,18 resulting in enhanced association and phosphorylation of PTEN by LKB1, which increased the phosphorylation, stability, and activation of PTEN in vitro and in vivo. Thus, our results suggest that diabetes mellitus, via hyperglycemia-driven ONOO–, resulted in accelerated apoptosis by LKB1-mediated but PTEN-dependent Akt inhibition.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
A full description of materials and methods used, including Akt activity assay, small interfering RNA (siRNA) gene silencing, measurement of endogenous ONOO–, PTEN activity assay, in vitro kinase assays, quantitative detection of DNA fragmentation by ELISA, and immunohistochemistry, can be found in the online-only Data Supplement.

Cell Culture and Treatment With ONOO– and Insulin
Human umbilical vein endothelial cells (HUVECs) and endothelial cell culture medium were purchased from Cascade Biologics (Portland, Ore). HUVECs were maintained in Medium 200 supplemented with a low-serum-growth supplement kit, penicillin (100 U/mL), and streptomycin (100 µg/mL). The concentrations of ONOO– were determined spectrophotometrically in 0.1 mol/L NaOH ({epsilon}302=1670 mol · L–1 · s–1). To avoid a pH shift, ONOO– was diluted in 0.1 mol/L NaOH. After serum starvation for 6 hours, HUVECs were treated with 5 µmol/L ONOO– as described previously.19 Thirty-five minutes after ONOO– treatment, HUVECs were stimulated with insulin (100 nmol/L) for 10 minutes.

High-Glucose Treatment of HUVECs
After reaching confluence, HUVECs were exposed to normal glucose (NG; 5 mmol/L D-glucose), high glucose (HG; 30 mmol/L D-glucose), or hyperosmotic control (5 mmol/L D-glucose plus 25 mmol/L L-glucose) for up to 72 hours with a daily change of culture media. When needed, pharmacological inhibitors, including uric acid (0.5 mmol/L), Tempol (0.05 mmol/L), or NG-nitro-L-arginine methyl ester (L-NAME; 0.5 mmol/L), were preincubated 1 hour before HG. For siRNA experiments, control or specific siRNA was added to the HUVECs and kept for the time indicated.

Streptozotocin-Induced Diabetes Mellitus
After 4 hours of fasting, 10-week-old male mice were made diabetic by injection of streptozotocin (STZ; 50 mg/kg of body weight daily) for 5 consecutive days.20 Diabetes mellitus is defined as random blood glucose levels of >300 mg/dL. Control and STZ-injected mice were given Tempol (44 mg/kg body weight daily) in their drinking water for 28 days21 or insulin injections (2.5 U/kg body weight twice daily). STZ animals also were injected retro-orbitally with either mouse-specific PTEN siRNA or control siRNA (200 µL; final dose, 1 mg/kg) every 3 days for 6 days using in vivo jetPEI (Polyplus-Transfection, Illkirch, France) according to the manufacturer’s recommendations. At the time indicated, mice were euthanized by isoflurane inhalation, and aortas were removed and immediately frozen in liquid nitrogen or fixed in 4% paraformaldehyde. For further details, see the online-only Data Supplement.

Statistical Analysis
Values are presented as mean±SD. Differences between experimental groups were determined by 1-way or 2-way ANOVA, followed by Student t test as appropriate. An unpaired Student t test was performed for single comparisons between groups. Values of P<0.05 were considered statistically significant.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
ONOO– Inhibits Akt-Ser473 Phosphorylation and Activity
To determine the effects of ONOO– on the insulin signaling cascade, we first investigated the effects of ONOO– on Akt-Ser473 phosphorylation and Akt activity in cultured HUVECs. Confluent HUVECs were treated with insulin (100 nmol/L) with or without ONOO–. As expected, insulin significantly increased the phosphorylation of Akt-Ser473 and Akt activity (Figure 1A) as measured by Akt-dependent GSK-3β fusion protein phosphorylation in in vitro assays. Exposure of HUVECs to pathologically relevant concentrations of ONOO– (5 µmol/L) significantly attenuated the basal and insulin-stimulated Akt-Ser473 phosphorylation (70% and 76% reduction; P<0.01) and Akt activity (80% and 88% reduction; P<0.01) (Figure 1A).


Figure 1186427
View larger version (37K):
[in this window]
[in a new window]

  		Figure 1. ONOO– inhibits insulin-, VEGF-, or IGF-stimulated PI-3 kinase signaling. Confluent HUVECs were treated with ONOO– (5 µmol/L) for 5 minutes before being exposed to insulin (100 nmol/L) for 10 minutes. Cells were then harvested for detection of (A) Akt phosphorylation and activity, (B) PDK1 phosphorylation (n=6; 2-way ANOVA: {clubsuit}P<0.01 vs control; {dagger}P<0.01 vs control; #P<0.01 vs insulin; P>0.5, ONOO– vs ONOO–/insulin), (C) inhibition of VEGF- (50 ng/mL, 10 minutes) or IGF- (50 ng/mL, 10 minutes) enhanced Akt phosphorylation and Akt activity, and (D) PDK1 phosphorylation by ONOO–. The blot is a representative of 3 blots from 3 independent experiments (n=3: 1-way ANOVA: {clubsuit}P<0.01 vs control; {dagger}P<0.01, ONOO–/VEGF vs VEGF, ONOO–/IGF vs IGF).

We next determined whether ONOO– altered the phosphorylation of PDK1, PI3K, and Akt. The phosphorylation of PDK1 at Ser241 is reported to correlate with its activity.22 As expected, insulin dramatically increased the Ser241 phosphorylation of PDK1 in HUVECs (Figure 1B). However, exposure of HUVECs to ONOO– (5 µmol/L) markedly diminished insulin-enhanced Ser241 phosphorylation of PDK1.

Because a decrease in PDK-1 and Akt might be due to inhibition of insulin receptor substrate 1 (IRS)-1–dependent PI3K activation, we next determined whether ONOO– altered the Ser307 phosphorylation of IRS-1, which is reported to negatively modulate IRS-1–associated PI3K activity.23 Unexpectedly, ONOO– inhibited the Ser307 phosphorylation of IRS-1 in HUVECs (data not shown), suggesting that the effects of ONOO– on both PDK-1 and Akt were not from decreased activation of PI3K by IRS-1.

We next determined whether ONOO– altered the phosphorylation of both PDK-1 and Akt enhanced by growth factors such as vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF). As shown in Figure 1C, both VEGF and IGF significantly increased the Ser473 phosphorylation of Akt and Akt activity. The addition of ONOO– significantly ablated VEGF- or IGF-enhanced Akt-Ser473 phosphorylation or Akt activity (Figure 1C). Concomitantly, VEGF or IGF significantly increased the phosphorylation of PDK1, whereas ONOO– (5 µmol/L) abolished VEGF- and IGF-stimulated PDK1 phosphorylation (Figure 1D). Taken together, our results suggest that ONOO– inhibited both PDK-1 and Akt, likely via the suppression of the PI3K pathway.

Inhibition of Akt by ONOO– Is PTEN Dependent
The phosphorylation of both PDK1 and Akt is concomitantly controlled by levels of phosphatidylinositol-3,4,5-P3, which is regulated by PI3K and PTEN.8 PTEN is a PtdIns-3,4,5-P3 D3-phosphatase that inhibits both PDK and Akt signaling by dephosphorylating phosphatidylinositol-3,4,5-P3.24 Phosphorylation of PTEN at Ser380/Thr382/383 is essential for its stability because it prevents degradation.25,26 Therefore, we examined the effects of ONOO– on the levels of p-PTEN-S380/T382/383 and total PTEN. As shown in Figure 2A, ONOO– (5 µmol/L) significantly increased the levels of phosphorylated PTEN (S380/T382/383) by 1.5-fold. Similarly, compared with HUVECs treated with vehicles, ONOO– increased the levels of PTEN by 40% (P<0.05; Figure 2B). Concomitantly, ONOO– increased PTEN activity by 2-fold under basal and insulin-stimulated conditions (Figure 2C).


Figure 2186427
View larger version (43K):
[in this window]
[in a new window]

  		Figure 2. ONOO–-induced Akt inhibition is PTEN dependent. ONOO– increases (A) p-PTEN-S380/T382/383, (B) PTEN level, and (C) PTEN activity (n=6; 2-way ANOVA: {dagger}P<0.01 vs control; {clubsuit}P<0.01 vs insulin). D, HUVECs were transfected with either control siRNA or PTEN siRNA (100 nmol/L) for 48 hours and treated with ONOO– (5 µmol/L) for 45 minutes. The blot is a representative of 4 blots obtained from 4 separate experiments. Notably, PTEN siRNA abolishes ONOO–-induced inhibition of Akt (n=4; 1-way ANOVA: {clubsuit}P<0.01, PTEN siRNA vs control; {dagger}P<0.01, ONOO–/control siRNA vs control siRNA; #P<0.01, ONOO–/PTEN siRNA vs ONOO–/control siRNA).

To determine whether PTEN was responsible for ONOO–-induced Akt inhibition, HUVECs were transfected with PTEN-specific siRNA or control siRNA. As shown in Figure 2D, transfection of siRNA but not control siRNA significantly suppressed both endogenous PTEN and PTEN phosphorylation in HUVECs. However, neither PTEN-specific siRNA nor control siRNA altered the levels of Akt-Ser473 phosphorylation or Akt activity (Figure 2D). PTEN-specific siRNA but not control siRNA reversed ONOO–-induced inhibition on both Akt phosphorylation and Akt activity in HUVECs. In parallel, PTEN siRNA but not control siRNA abolished ONOO–-enhanced PTEN and PTEN phosphorylation in HUVECs (Figure 2D). Concomitantly, transfection of PTEN siRNA but not control siRNA abolished ONOO–-suppressed PDK1 phosphorylation in HUVECs (Figure 2D). Taken together, these results imply that ONOO–-induced Akt inhibition is PTEN-mediated.

PTEN-Dependent Akt Inhibition by ONOO– Is LKB1 Mediated
We previously demonstrated that ONOO– significantly increases Ser428 phosphorylation of the serine/threonine kinase LKB1 in bovine aortic endothelial cells.27 Moreover, evidence exists that LKB1 phosphorylates PTEN in cancer cells;28 however, LKB1 phosphorylation site(s) had not been identified. We further assayed whether increased PTEN phosphorylation was mediated by LKB1 in HUVECs and, if so, the phosphorylation site(s) of PTEN by LKB1. As depicted in Figure 3A, ONOO– significantly increased both basal and insulin-treated LKB1-Ser428 phosphorylation by 60% (P<0.01). Although LKB1 is located primarily in the nucleus,28 ONOO– significantly increased the translocation of LKB1 from the nucleus to the cytoplasm and plasma membrane (Figure 3B). In contrast, PTEN was located mainly in cytosols in unstimulated HUVECs. Exposure of HUVECs to ONOO– increased membrane-associated PTEN (Figure 3C).


Figure 3186427
View larger version (31K):
[in this window]
[in a new window]

  		Figure 3. LKB1 is required for ONOO–-induced Akt inhibition. A, ONOO– enhances phosphorylation of LKB1 at Ser428 (n=6; 2-way ANOVA: {dagger}P<0.01, ONOO– vs control; {clubsuit}P<0.05 vs insulin). No significance existed between ONOO– and ONOO– plus insulin-induced LKB1 phosphorylation. B, ONOO– increases the translocation of LKB1 from the nucleus to the cytoplasm and membrane (n=4; unpaired t test: {clubsuit}P<0.01, ONOO– vs control; {dagger}P<0.05, ONOO– vs control). Nuclear, cytosolic, and membrane fractions were isolated as described in the online-only Data Supplement. LKB1 or PTEN was detected in Western blots using the specific antibody. C, ONOO– enhances membrane-bound LKB1 and PTEN. The photo is a representative of 4 individual experiments by confocal microscopy. D, HUVECs were transfected with control siRNA or LKB1 siRNA (100 nmol/L) for 48 hours and treated with ONOO– (5 µmol/L) for 45 minutes. The blot is a representative blot from 4 independent experiments. Notably, LKB1 siRNA but not control siRNA attenuates ONOO–-induced PTEN phosphorylation and Akt inhibition (n=4; 1-way ANOVA: {dagger}P<0.01, LKB1 siRNA vs control; #P<0.01, ONOO– vs control; {clubsuit}P<0.01, LKB1 siRNA/ONOO– vs control siRNA/ONOO–).

To further examine the role of LKB1 in ONOO–-induced, PTEN-mediated Akt inhibition, HUVECs were transfected with LKB1 siRNA or control siRNA. Transfection of LKB1-specific siRNA but not control siRNA reduced LKB1 by 80% (Figure 3D). Interestingly, LKB1 siRNA but not control siRNA abolished ONOO–-enhanced PTEN phosphorylation at Ser380/Thr382/383 and AMPK phosphorylation in HUVECs (Figure 3D). Furthermore, LKB1 siRNA abolished ONOO–-enhanced inhibition of Akt phosphorylation, whereas control siRNA had no effect (Figure 3D).

We next investigated whether ONOO– increased the interaction of LKB1-PTEN. LKB1 was immunoprecipitated and Western blotted with the antibody against PTEN or vice versa. Compared with a weak association of LKB1 with PTEN in control cells, ONOO– significantly increased the association of LKB1 with PTEN (Figure 4A and 4B).


Figure 4186427
View larger version (26K):
[in this window]
[in a new window]

  		Figure 4. ONOO– enhances LKB1 and PTEN interactions. A and B, ONOO– increases the association of LKB1 and PTEN. LKB1 or PTEN was immunoprecipitated, and PTEN or LKB1 was detected by Western blotting (n=4; 2-way ANOVA; {dagger}P<0.01, ONOO– vs control; {clubsuit}P<0.01, ONOO–/insulin vs insulin). C, Recombinant LKB1 enhances Akt-Ser-473 phosphorylation and activity in vitro. GSK-3 fusion protein was incubated with kinase buffer alone (lane 1), recombinant Akt1 (lane 2), recombinant LKB1 (lane 3), recombinant Akt1 and LKB1 (lane 4), or recombinant Akt1, LKB1, and PTEN (lane 5) in the presence of ATP. Reactions were allowed to proceed for 30 minutes at 37°C (n=3; 1-way ANOVA: {clubsuit}P<0.01, Akt1+LKB1+GSK3β or +PTEN vs Akt1+GSK3β). D, LKB1 phosphorylates recombinant human PTEN at Ser380/Thr382/383. Recombinant PTEN was incubated with either kinase buffer alone (lane 1) or recombinant LKB1 (lane 2) in the presence of ATP for 30 minutes at 37°C. Representative Western blots for phosphorylated PTEN-S380/T382/383 (top) and total PTEN (bottom) are shown (n=6; unpaired t test: {clubsuit}P<0.01 vs PTEN alone).

LKB1 Phosphorylates PTEN at Ser380/Thr382/383
Phosphorylation of PTEN at Ser380/Thr382/383 within its C-terminal tail is reported to increase its stability.25,26 Because ONOO– increased the association of PTEN with LKB1, we first determined whether LKB1 phosphorylated PTEN in vitro. Incubation of recombinant LKB1 with recombinant Akt1 in vitro increased Akt1 phosphorylation and activity (Figure 4C, lane 4 versus lane 2), excluding a direct inhibition of Akt1 or GSK-3β by LKB1. In addition, exogenous PTEN had no effect on Akt activity (Figure 4C, lane 5 versus lane 4), implying that PTEN did not act as a protein phosphatase to inhibit Akt. Furthermore, LKB1 significantly increased the phosphorylation of PTEN at Ser380/Thr382/383 (Figure 4D, lane 2). Because neither LKB1 nor PTEN inhibited the phosphorylation of Akt or GSK-3β in vitro, the inhibitory effects of ONOO– on Akt/PDK1 in HUVECs are likely via a PTEN-dependent degradation of lipid molecules [PtdIns(3,4,5)P3] of PI3K. Because ONOO– increased PTEN phosphorylation at Ser380/Thr382/383, resulting in an increased PTEN half-life, increased association of LKB1 with PTEN might be responsible for ONOO–-enhanced PTEN phosphorylation and membrane association.29,30

HG Induces PTEN-Dependent Endothelial Cell Apoptosis in HUVECs
Both diabetes mellitus and hyperglycemia are known to increase ONOO–, as assayed by 3-nitrotyrosine (3-NY), a footprint of ONOO– in cultured cells.15,16 We next determined whether HG, via ONOO–, altered the LKB1-PTEN-Akt axis in HUVECs. Consistent with earlier reports,31,32 exposure of HUVECs to HG (30 mmol/L) for 72 hours inhibited Akt Ser473 phosphorylation by 80% (P<0.01) (Figure 5A); increased the levels of both PTEN and phosphorylated PTEN (S380/T382/383) by 50% and 70%, respectively (P<0.01) (Figure 5A and 5D); and increased the Ser428 phosphorylation of LKB1 (Figure 5D). Importantly, transfection of PTEN siRNA but not control siRNA, which did not alter LKB1 phosphorylation, abolished HG-induced inhibition of Akt phosphorylation (Figure 5A). These data suggest that LKB1 might be an upstream kinase for PTEN in HUVECs.


Figure 5186427
View larger version (44K):
[in this window]
[in a new window]

  		Figure 5. Akt inhibition and apoptosis induced by high glucose via ONOO– in HUVECs are dependent on PTEN. Exposure of HUVECs to HG (30 mmol/L) decreases Akt phosphorylation but increases PTEN levels (A) and apoptosis (B), measured as DNA fragmentation (n=6; 1-way ANOVA: {clubsuit}P<0.01 vs NG; {dagger}P<0.01, HG/S-PTEN vs HG). Transfection of HUVECs with PTEN siRNA (100 nmol/L) completely abolished the effects of HG on Akt. C and D, HG-enhanced ONOO–-dependent dihydrorhodamine 123 (DHR) oxidation, LKB1-S428, PTEN-S380/T382/383 phosphorylation, and PTEN are attenuated by uric acid (UA; 0.5 mmol/L), the ONOO– scavenger Tempol (TMP; 0.05 mmol/L), an SOD mimetic, or L-NAME (0.5 mmol/L), an NO synthase inhibitor (n=4; 1-way ANOVA: {clubsuit}P<0.05 vs NG; {dagger}P<0.01, HG+inhibitors vs HG). NG indicates normal glucose (5 mmol/L D-glucose); OG, osmotic control glucose (5 mmol/L D-glucose+25 mmol/L L-glucose); HG, 30 mmol/L D-glucose; S-Ctrl, control siRNA; and S-PTEN, PTEN siRNA.

Both LKB1 and PTEN are tumor suppressors, whereas Akt functions as a survival factor. Thus, an increase in the first 2 and/or suppression of the last by ONOO– are expected to cause apoptosis. Indeed, HG significantly reduced the cell viability, as assayed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, and increased endothelial cell apoptosis by 130%, as measured by DNA fragmentation (Figure 5B). As expected, PTEN siRNA abrogated the HG-induced apoptosis in HUVECs, whereas control siRNA had no effect (Figure 5B).

HG Via ONOO– Causes the Upregulation of LKB1 and PTEN in HUVECs
To assay the formation of ONOO–, we measured HG-induced dihydrorhodamine 123 oxidation in HUVECs. Earlier studies suggest that dihydrorhodamine 123 reacts with ONOO– but not with its precursor, O2•– or NO.33 Compared with normal cells, HG but not hyperosmotic control glucose significantly increased the oxidation of dihydrorhodamine 123 (Figure 5C), implying that HG increased ONOO–. Concomitantly, HG but not hyperosmotic control glucose significantly increased 3-NY, a footprint in cultured endothelial cells (P.S., J.X., and M.-H.Z., unpublished data, 2007). Furthermore, administration of either an O2•– scavenger, Tempol, or an NO synthase inhibitor, L-NAME, both of which prevent ONOO– formation, like ONOO– scavenger uric acid, abolished HG-induced dihydrorhodamine oxidation. Because uric acid, Tempol, and L-NAME alone had no effect on basal dihydrorhodamine oxidation (data not shown), these data suggest that HG significantly increased the formation of ONOO– in HUVECs.

We next determined whether ONOO– generated by HG was involved in HG-induced PTEN-mediated Akt inhibition. Because ONOO– has a half-life of <1 second at physiological pH 7.434 and ONOO– can initiate both nitrosative and oxidative reactions in both in vitro and in vivo,12,13 scavenging of O2•– or inhibition of NO with L-NAME was used to obtain indirect evidence for ONOO– in HUVECs. As shown in Figure 5C and 5D, either scavenging O2•– with Tempol (0.05 mmol/L), a superoxide dismutase (SOD) mimetic, or L-NAME (0.5 mmol/L), a nonselective NO synthase inhibitor, abolished HG-enhanced Ser428 phosphorylation of LKB1 and the upregulation of PTEN in HUVECs. Furthermore, uric acid (0.5 mmol/L), a potent scavenger for ONOO–, had the same effects. Because none of these reagents altered the basal levels of LKB1 and PTEN in HUVECs (data not shown), these results implied that reactive nitrogen species, likely ONOO–, might play a causal role in the upregulation of LKB1 and PTEN caused by HG exposure in HUVECs.

Inhibitory Effects of Hyperglycemia on Akt Are PTEN Dependent in Diabetic Mice
We further investigated the upregulation of LKB1 and PTEN and accelerated apoptosis in diabetes mellitus in vivo. Diabetes mellitus was induced in mice by STZ injection, and mice were concomitantly treated with an SOD mimetic, Tempol, or insulin. Injection of STZ significantly increased serum glucose (460±20 mg/dL) compared with the mice with vehicle (180±15 mg/dL; n=5; P<0.01). Administration of insulin markedly reduced the glucose to levels (185±19 mg/dL) similar to those seen in vehicle. Treatment with Tempol for 4 weeks did not alter the blood glucose levels of the control mice (188±16 mg/dL; n=5; P>0.5) or the STZ-induced diabetic mice (465±23 mg/dL; n=5; P>0.5). No significant difference in body weight was observed among these groups (data not shown).

We first determined whether diabetes mellitus increased ONOO– in vivo. 3-NY, a stable marker for ONOO–, was measured in aortic homogenates in Western blots. As shown in Figure 6A, diabetes mellitus significantly increased the levels of 3-NY–positive proteins. Both insulin and Tempol treatments alone significantly attenuated the diabetes-enhanced 3-NY (Figure 6A) whereas neither PTEN-siRNA nor control siRNA altered the levels of 3-NY (Figure 6A). In parallel with 3-NY reduction, administration of insulin or Tempol or PTEN-specific siRNA significantly attenuated diabetes-enhanced DNA fragmentation in mouse aortic homogenates, suggesting that diabetes mellitus via hyperglycemia-driven reactive oxygen and nitrogen species caused accelerated apoptosis.


Figure 6186427
View larger version (39K):
[in this window]
[in a new window]

  		Figure 6. PI3K/Akt signaling impaired by diabetes mellitus via ONOO– is PTEN dependent in vivo. Mice injected with vehicle or STZ to induce diabetes mellitus were concomitantly treated with Tempol or insulin (2.5 U/kg) as described in Methods. The aortic homogenates were subjected to the measurement of (A) 3-NY–positive proteins and apoptosis (measured by DNA fragmentation), (B) LKB1 Ser428 phosphorylation, (C) phosphorylated PTEN(S380/T382/383) and PTEN levels, and (D) Akt Ser473 phosphorylation (n=5; 1-way ANOVA: {clubsuit}P<0.01, STZ, STZ/S-Ctrl, or STZ/S-PTEN vs vehicle; {dagger}P<0.01 STZ/insulin, STZ-S-PTEN, or STZ/Tempol vs STZ). S-Ctrl indicates control siRNA; S-PTEN, PTEN siRNA.

We next determined whether LKB1-dependent PTEN-mediated Akt inhibition was operated in vivo. As shown in Figure 6B, STZ injection significantly increased LKB1 phosphorylation at Ser428. Administration of insulin or Tempol abolished STZ-induced LKB1 phosphorylation (Figure 6B). In parallel, STZ injection significantly increased the PTEN phosphorylation and PTEN levels, which was sensitive to insulin or Tempol (Figure 6C). Interestingly, administration of PTEN siRNA suppressed diabetes-enhanced PTEN phosphorylation and PTEN levels (Figure 6C) but did not alter the phosphorylation of LKB1 (Figure 6B), suggesting that LKB1 might be an upstream kinase of PTEN. Moreover, administration of either PTEN siRNA or insulin or Tempol abolished diabetes-induced Akt inhibition (Figure 6D), suggesting that diabetes mellitus via reactive oxygen or nitrogen species suppressed Akt via an LKB1-dependent PTEN-mediated manner in vivo.

We next investigated the changes in endothelium caused by diabetes mellitus. As shown in Figure 7, diabetes mellitus significantly increased 3-NY, PTEN, and apoptosis, localized mainly in endothelium (Figure 7B, 7H, and 7N). Interestingly, insulin, which lowered STZ-induced hyperglycemia, reduced STZ-enhanced 3-NY, PTEN, and apoptosis (Figure 7C, 7I, and 7O). Similarly, administration of Tempol in STZ-injected mice also reversed the effects of diabetes mellitus on 3-NY, PTEN, and apoptosis (Figure 7F, 7L, and 7R). Administration of PTEN-specific siRNA but not control siRNA lowered the levels of PTEN in the endothelium of aortic rings by 70% (Figure 7K and 7J) and significantly reduced diabetes-enhanced apoptosis (Figure 7Q) without altering diabetes-enhanced 3-NY (Figure 7E). Taken together, these data imply that diabetes mellitus, via hyperglycemia-driven ONOO–, upregulated PTEN, resulting in accelerated apoptosis in endothelium.


Figure 7186427
View larger version (33K):
[in this window]
[in a new window]

  		Figure 7. ONOO– generation, PTEN upregulation, and single-strand DNA breakage in diabetic blood vessels. A through F, Immunohistochemical staining for 3-NY (brown) in vehicle rings (A), in rings from STZ mice (B), in STZ mice treated with insulin (C), in STZ mice injected with control siRNA (D), in mice injected with PTEN siRNA (E), or in mice fed Tempol (F). G through L, Immunohistochemical staining for PTEN (red). M through R, Terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) (red fluorescent), an indicator of DNA-strand breakage. Magnification x200. Similar immunohistochemical profiles were seen in 5 to 6 aorta rings per group. S-Ctrl indicates control siRNA; S-PTEN, PTEN siRNA.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we have presented evidence that hyperglycemia via ONOO– can significantly elevate levels of PTEN, which appears to mediate the inhibitory effects of HG on PI3K/Akt signaling in vascular endothelium. We also have demonstrated that LKB1 was required for ONOO–-induced Akt inhibition. Our data support the hypothesis that ONOO– production associated with HG triggers LKB1-dependent activation of PTEN, which suppresses Akt signaling and enhances apoptosis in both cultured HUVECs and vascular endothelium of the aortas from diabetic mice (Figure 8). Thus, our results unveil a novel mechanism by which diabetes mellitus may cause vascular endothelial dysfunction and cell death.


Figure 8186427
View larger version (15K):
[in this window]
[in a new window]

  		Figure 8. Proposed mechanism for diabetes-enhanced apoptosis in endothelial cells. STZ-induced hyperglycemia enhances LKB1 and PTEN phosphorylation, PTEN levels, and consequent inhibition of Akt with resultant apoptosis. Inhibition of ONOO– formation, by inhibition of either its constituent O2•– via the SOD mimetic Tempol or nitric oxide by L-NAME or its scavenger uric acid, blocks hyperglycemia-augmented PTEN and concomitant Akt inhibition. Overall, we consider in diabetes mellitus that hyperglycemia via ONOO– increases apoptosis via an LKB1-mediated PTEN-dependent Akt inhibition.

Several lines of evidence are consistent with the hypothesis that hyperglycemia, via ONOO–, enhances PTEN stability, which suppresses PI3K-dependent phosphorylation of Akt. First, HG significantly increased ONOO– in HUVECs, as assayed by dihydrorhodamine 123 oxidation and 3-NY–positive proteins. Second, ONOO– or HG treatment of HUVECs significantly inhibited both basal and insulin-stimulated phosphorylation of Akt-Ser473 and Akt activity. Third, inhibition of PTEN with siRNA but not with control siRNA prevented ONOO– or HG-induced Akt inhibition and apoptosis. Consistent with these findings, diabetic mice exhibited a parallel reduction in Ser473 phosphorylation of Akt in the aortas, along with a marked increase in PTEN. Fourth, administration of insulin, which lowered blood serum glucose to levels comparable to those in nondiabetic controls, attenuated diabetes-enhanced PTEN and Ser428 phosphorylation of LKB1. These results suggest that hyperglycemia is responsible for diabetes-enhanced PTEN upregulation. Importantly, insulin treatment also ablated 3-NY staining in diabetes mellitus, suggesting that hyperglycemia might be a driving factor for ONOO–. Fifth, elevated LKB1 and PTEN phosphorylation and PTEN levels induced by HG were significantly abolished by either the inhibition of ONOO– formation (Tempol and L-NAME) or the ONOO– scavenger, uric acid. Finally, administration of Tempol, an SOD mimetic, which suppressed ONOO– formation as evidenced by decreased 3-NY staining, increased Akt phosphorylation but suppressed apoptosis in parallel with decreased levels of PTEN and LKB1 phosphorylation in diabetic mice, supporting a potential role of ONOO– in diabetes mellitus. Taken together, our results indicate that STZ-induced hyperglycemia inhibited PI3K/Akt by upregulating PTEN via increased stability.

We provided further evidence that the tumor suppressor LKB1 might play an important role in the development of vascular injury and insulin resistance in the vasculature. Previous studies have demonstrated that a skeletal muscle-selective knockout of LKB1 can increase insulin sensitivity,35 and several studies have suggested that LKB1 may interact with PTEN to suppress tumor growth.28,36 Our data reveal that LKB1 significantly increased the phosphorylation of PTEN in vitro at S380/T382/T383 sites known for prolonged stability of PTEN. Our findings are consistent with the idea that LKB1 increases the stability of PTEN by phosphorylating PTEN at Ser380/Thr382/383. First, Ser428 LKB1 phosphorylation in response to ONOO– or hyperglycemia was accompanied by increased PTEN phosphorylation. Second, ONOO– notably augmented the translocation of LKB1 from the nucleus to the cytoplasm and cytoplasmic membranes. Third, knockdown of LKB1 with LKB1-specific siRNA clearly normalized phosphorylated PTEN and restored Akt phosphorylation inhibited by ONOO–. Fourth, LKB1 and PTEN interactions, which were weak under control or insulin-treated conditions, were significantly enhanced after ONOO– treatment. Finally, in STZ-induced diabetic mice, increases in aortic levels of PTEN, Ser428-phosphorylated LKB1, and 3-NY occurred in parallel with a reduction in Ser473 Akt phosphorylation. To the best of our knowledge, this is the first direct evidence that ONOO– augments the functional link between LKB1 and PTEN.

Recent evidence indicates that diabetes mellitus and hyperglycemia cause oxidant stress.37 Endothelial cells are capable of generating ONOO– because of their capacity to simultaneously produce O2•– and NO.38,39 Previous studies have demonstrated that HG augments O2•– release,4,40,41 which traps the vasorelaxant NO, leading to increased ONOO– levels in endothelial cells.15 Recent clinical data found that 3-NY staining was associated with increased apoptosis in diabetes mellitus, suggesting a correlation between apoptosis and ONOO– generation. For example, Frustaci et al1 found that apoptosis increased 61- and 85- fold in endothelial cells and cardiomyocytes, respectively, in ventricular myocardial biopsies from diabetic humans. However, these studies did not establish a causal role of ONOO– in diabetic apoptosis. In the present study, we have for the first time demonstrated that ONOO–-dependent PTEN caused apoptosis in diabetes mellitus. However, our results might be applied only to cells or tissues when NO is present because ONOO– formation requires NO. In the absence of NO, O2•– might become hydrogen peroxide, which, unlike ONOO–, causes oxidative inactivation as a result of disulfide bond formation in PTEN.42–44

Recently, it has been shown that upregulation of PTEN is involved in the inhibitory effects of resistin and free fatty acids on insulin signaling in endothelial cells.10,11 Indeed, Nakashima et al45 demonstrated that overexpression of PTEN in 3T3-L1 cells inhibits glucose uptake and GLUT4 translocation in vitro, whereas microinjection of a PTEN antibody increases basal and insulin-stimulated GLUT4 translocation in vitro. Tissue-specific deletion of PTEN in liver,46 muscle,9 and adipose tissue47 results in insulin hypersensitivity. More interestingly, systemic administration of PTEN antisense oligonucleotides once a week for 4 weeks can reverse hyperglycemia in db/db (mutations in the gene that encodes the receptor for leptin) and ob/ob diabetic mice.48 Thus, our observations may hold importance for insulin resistance in type 2 diabetes mellitus. Although we have shown that administration of PTEN siRNA for 6 days can completely restore Akt phosphorylation in mice aortas, this treatment did not reverse hyperglycemia. Nevertheless, our experimental paradigm in endothelial cells suggests that PTEN may be a promising target for therapeutic intervention for diabetic complications. In the insulin-resistant state, the metabolic IRS/PI3K/PDK/Akt branch becomes insensitive to insulin stimulation. More importantly, in the presence of defective insulin-stimulated glucose uptake, a defect in insulin-induced endothelium-dependent vasodilation also exists, suggesting a systemic deregulation of the PI3K/Akt pathway responsible for changes in insulin-regulated metabolism and cardiovascular functions. Our results show that hyperglycemia can increase PTEN levels and that PTEN-specific siRNA can reverse the inhibitory effects of hyperglycemia on Akt activation. These results suggest that the upregulated PTEN may mediate the inhibitory effects of hyperglycemia on insulin signaling and endothelial NO synthase activation in endothelial cells. Because PTEN plays an essential role in maintaining cell survival/death signals, upregulation of PTEN by ONOO–-dependent LKB1 may provide a link between oxidative stress and endothelial injury, an early phenomenon observed in type 1 and 2 diabetes mellitus.


*    Acknowledgments
 
Sources of Funding

This work was supported by National Institutes of Health grants (HL079584, HL074399, and HL080499), a research award from the American Diabetes Association, a research award from the Juvenile Diabetes Research Foundation, a research award from the Oklahoma Center for Advancement of Science and Technology, and the Travis Endowed Chair in Endocrinology, University of Oklahoma Health Sciences Center.

Disclosures

None.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Frustaci A, Kajstura J, Chimenti C, Jakoniuk I, Leri A, Maseri A, Nadal-Ginard B, Anversa P. Myocardial cell death in human diabetes. Circ Res. 2000; 87: 1123–1132.[Abstract/Free Full Text]
  2. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33): UK Prospective Diabetes Study (UKPDS) Group. Lancet. 1998; 352: 837–853.[CrossRef][Medline] [Order article via Infotrieve]
  3. Nathan DM, Lachin J, Cleary P, Orchard T, Brillon DJ, Backlund JY, O’Leary DH, Genuth S. Intensive diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus. N Engl J Med. 2003; 348: 2294–2303.[Abstract/Free Full Text]
  4. Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci U S A. 2000; 97: 12222–12226.[Abstract/Free Full Text]
  5. Scherrer U, Randin D, Vollenweider P, Vollenweider L, Nicod P. Nitric oxide release accounts for insulin’s vascular effects in humans. J Clin Invest. 1994; 94: 2511–2515.[Medline] [Order article via Infotrieve]
  6. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science. 1997; 275: 1943–1947.[Abstract/Free Full Text]
  7. Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, Ruland J, Penninger JM, Siderovski DP, Mak TW. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell. 1998; 95: 29–39.[CrossRef][Medline] [Order article via Infotrieve]
  8. Cantley LC, Neel BG. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A. 1999; 96: 4240–4245.[Abstract/Free Full Text]
  9. Wijesekara N, Konrad D, Eweida M, Jefferies C, Liadis N, Giacca A, Crackower M, Suzuki A, Mak TW, Kahn CR, Klip A, Woo M. Muscle-specific PTEN deletion protects against insulin resistance and diabetes. Mol Cell Biol. 2005; 25: 1135–1145.[Abstract/Free Full Text]
  10. Wang XL, Zhang L, Youker K, Zhang MX, Wang J, Lemaire SA, Coselli JS, Shen YH. Free fatty acids inhibit insulin signaling-stimulated endothelial nitric oxide synthase activation through upregulating PTEN or inhibiting Akt kinase. Diabetes. 2006; 55: 2301–2310.[Abstract/Free Full Text]
  11. Shen YH, Zhang L, Gan Y, Wang X, Wang J, LeMaire SA, Coselli JS, Wang XL. Up-regulation of PTEN (phosphatase and tensin homolog deleted on chromosome ten) mediates p38 MAPK stress signal-induced inhibition of insulin signaling: a cross-talk between stress signaling and insulin signaling in resistin-treated human endothelial cells. J Biol Chem. 2006; 281: 7727–7736.[Abstract/Free Full Text]
  12. Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium. 2004; 11: 89–97.[CrossRef][Medline] [Order article via Infotrieve]
  13. Radi R. Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci U S A. 2004; 101: 4003–4008.[Abstract/Free Full Text]
  14. Zou MH, Shi C, Cohen RA. Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest. 2002; 109: 817–826.[CrossRef][Medline] [Order article via Infotrieve]
  15. Zou MH, Shi C, Cohen RA. High glucose via peroxynitrite causes tyrosine nitration and inactivation of prostacyclin synthase that is associated with thromboxane/prostaglandin H(2) receptor-mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells. Diabetes. 2002; 51: 198–203.[Abstract/Free Full Text]
  16. Garcia Soriano F, Virag L, Jagtap P, Szabo E, Mabley JG, Liaudet L, Marton A, Hoyt DG, Murthy KG, Salzman AL, Southan GJ, Szabo C. Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation. Nat Med. 2001; 7: 108–113.[CrossRef][Medline] [Order article via Infotrieve]
  17. Zou MH, Hou XY, Shi CM, Nagata D, Walsh K, Cohen RA. Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase. J Biol Chem. 2002; 277: 32552–32557.[Abstract/Free Full Text]
  18. Hemminki A, Tomlinson I, Markie D, Jarvinen H, Sistonen P, Bjorkqvist AM, Knuutila S, Salovaara R, Bodmer W, Shibata D, de la Chapelle A, Aaltonen LA. Localization of a susceptibility locus for Peutz-Jeghers syndrome to 19p using comparative genomic hybridization and targeted linkage analysis. Nat Genet. 1997; 15: 87–90.[CrossRef][Medline] [Order article via Infotrieve]
  19. Zou MH, Hou XY, Shi CM, Kirkpatick S, Liu F, Goldman MH, Cohen RA. Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite. J Biol Chem. 2003; 278: 34003–34010.[Abstract/Free Full Text]
  20. O’Brien BA, Harmon BV, Cameron DP, Allan DJ. Beta-cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model. J Pathol. 1996; 178: 176–181.[CrossRef][Medline] [Order article via Infotrieve]
  21. Banday AA, Marwaha A, Tallam LS, Lokhandwala MF. Tempol reduces oxidative stress, improves insulin sensitivity, decreases renal dopamine D1 receptor hyperphosphorylation, and restores D1 receptor-G-protein coupling and function in obese Zucker rats. Diabetes. 2005; 54: 2219–2226.[Abstract/Free Full Text]
  22. Alessi DR, Deak M, Casamayor A, Caudwell FB, Morrice N, Norman DG, Gaffney P, Reese CB, MacDougall CN, Harbison D, Ashworth A, Bownes M. 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol. 1997; 7: 776–789.[CrossRef][Medline] [Order article via Infotrieve]
  23. Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF. Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J Biol Chem. 2002; 277: 1531–1537.[Abstract/Free Full Text]
  24. Lee JO, Yang H, Georgescu MM, Di Cristofano A, Maehama T, Shi Y, Dixon JE, Pandolfi P, Pavletich NP. Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell. 1999; 99: 323–334.[CrossRef][Medline] [Order article via Infotrieve]
  25. Vazquez F, Ramaswamy S, Nakamura N, Sellers WR. Phosphorylation of the PTEN tail regulates protein stability and function. Mol Cell Biol. 2000; 20: 5010–5018.[Abstract/Free Full Text]
  26. Torres J, Pulido R. The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus: implications for PTEN stability to proteasome-mediated degradation. J Biol Chem. 2001; 276: 993–998.[Abstract/Free Full Text]
  27. Xie Z, Dong Y, Zhang M, Cui MZ, Cohen RA, Riek U, Neumann D, Schlattner U, Zou MH. Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells. J Biol Chem. 2006; 281: 6366–6375.[Abstract/Free Full Text]
  28. Mehenni H, Lin-Marq N, Buchet-Poyau K, Reymond A, Collart MA, Picard D, Antonarakis SE. LKB1 interacts with and phosphorylates PTEN: a functional link between two proteins involved in cancer predisposing syndromes. Hum Mol Genet. 2005; 14: 2209–2219.[Abstract/Free Full Text]
  29. Vazquez F, Matsuoka S, Sellers WR, Yanagida T, Ueda M, Devreotes PN. Tumor suppressor PTEN acts through dynamic interaction with the plasma membrane. Proc Natl Acad Sci U S A. 2006; 103: 3633–3638.[Abstract/Free Full Text]
  30. Das S, Dixon JE, Cho W. Membrane-binding and activation mechanism of PTEN. Proc Natl Acad Sci U S A. 2003; 100: 7491–7496.[Abstract/Free Full Text]
  31. Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest. 2001; 108: 1341–1348.[CrossRef][Medline] [Order article via Infotrieve]
  32. Ido Y, Carling D, Ruderman N. Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes. 2002; 51: 159–167.[Abstract/Free Full Text]
  33. Kooy NW, Royall JA, Ischiropoulos H, Beckman JS. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic Biol Med. 1994; 16: 149–156.[CrossRef][Medline] [Order article via Infotrieve]
  34. Reiter CD, Teng RJ, Beckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem. 2000; 275: 32460–32466.[Abstract/Free Full Text]
  35. Koh HJ, Arnolds DE, Fujii N, Tran TT, Rogers MJ, Jessen N, Li Y, Liew CW, Ho RC, Hirshman MF, Kulkarni RN, Kahn CR, Goodyear LJ. Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3. Mol Cell Biol. 2006; 26: 8217–8227.[Abstract/Free Full Text]
  36. Jimenez AI, Fernandez P, Dominguez O, Dopazo A, Sanchez-Cespedes M. Growth and molecular profile of lung cancer cells expressing ectopic LKB1: down-regulation of the phosphatidylinositol 3'-phosphate kinase/PTEN pathway. Cancer Res. 2003; 63: 1382–1388.[Abstract/Free Full Text]
  37. Gutierrez J, Ballinger SW, Darley-Usmar VM, Landar A. Free radicals, mitochondria, and oxidized lipids: the emerging role in signal transduction in vascular cells. Circ Res. 2006; 99: 924–932.[Abstract/Free Full Text]
  38. van der Loo B, Labugger R, Skepper JN, Bachschmid M, Kilo J, Powell JM, Palacios-Callender M, Erusalimsky JD, Quaschning T, Malinski T, Gygi D, Ullrich V, Luscher TF. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med. 2000; 192: 1731–1744.[Abstract/Free Full Text]
  39. Dickhout JG, Hossain GS, Pozza LM, Zhou J, Lhotak S, Austin RC. Peroxynitrite causes endoplasmic reticulum stress and apoptosis in human vascular endothelium: implications in atherogenesis. Arterioscler Thromb Vasc Biol. 2005; 25: 2623–2629.[Abstract/Free Full Text]
  40. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, Hammes HP, Giardino I, Brownlee M. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature. 2000; 404: 787–790.[CrossRef][Medline] [Order article via Infotrieve]
  41. Cosentino F, Hishikawa K, Katusic ZS, Luscher TF. High glucose increases nitric oxide synthase expression and superoxide anion generation in human aortic endothelial cells. Circulation. 1997; 96: 25–28.[Abstract/Free Full Text]
  42. Lee SR, Yang KS, Kwon J, Lee C, Jeong W, Rhee SG. Reversible inactivation of the tumor suppressor PTEN by H2O2. J Biol Chem. 2002; 277: 20336–20342.[Abstract/Free Full Text]
  43. Leslie NR, Bennett D, Lindsay YE, Stewart H, Gray A, Downes CP. Redox regulation of PI 3-kinase signalling via inactivation of PTEN. EMBO J. 2003; 22: 5501–5510.[CrossRef][Medline] [Order article via Infotrieve]
  44. Connor KM, Subbaram S, Regan KJ, Nelson KK, Mazurkiewicz JE, Bartholomew PJ, Aplin AE, Tai YT, Aguirre-Ghiso J, Flores SC, Melendez JA. Mitochondrial H2O2 regulates the angiogenic phenotype via PTEN oxidation. J Biol Chem. 2005; 280: 16916–16924.[Abstract/Free Full Text]
  45. Nakashima N, Sharma PM, Imamura T, Bookstein R, Olefsky JM. The tumor suppressor PTEN negatively regulates insulin signaling in 3T3-L1 adipocytes. J Biol Chem. 2000; 275: 12889–12895.[Abstract/Free Full Text]
  46. Stiles B, Wang Y, Stahl A, Bassilian S, Lee WP, Kim YJ, Sherwin R, Devaskar S, Lesche R, Magnuson MA, Wu H. Liver-specific deletion of negative regulator PTEN results in fatty liver and insulin hypersensitivity [corrected]. Proc Natl Acad Sci U S A. 2004; 101: 2082–2087.[Abstract/Free Full Text]
  47. Kurlawalla-Martinez C, Stiles B, Wang Y, Devaskar SU, Kahn BB, Wu H. Insulin hypersensitivity and resistance to streptozotocin-induced diabetes in mice lacking PTEN in adipose tissue. Mol Cell Biol. 2005; 25: 2498–2510.[Abstract/Free Full Text]
  48. Butler M, McKay RA, Popoff IJ, Gaarde WA, Witchell D, Murray SF, Dean NM, Bhanot S, Monia BP. Specific inhibition of PTEN expression reverses hyperglycemia in diabetic mice. Diabetes. 2002; 51: 1028–1034.[Abstract/Free Full Text]
 

CLINICAL PERSPECTIVE

The major clinical manifestations of diabetes mellitus that result in most of the morbidity and mortality are results of its vascular pathology. The inner layers of vessels and capillaries are formed by endothelial cells. High blood sugar (glucose) alters the features of these cells by increasing the number of dead cells (apoptosis), which is regarded as an important factor in the pathogenesis of diabetes mellitus. PI-3 kinase/Akt has been shown to be associated with antiapoptosis, angiogenesis, and carcinogenesis. Our laboratory and others have demonstrated that PI-3 kinase/Akt, which is attenuated by high blood sugar, might play a causal role in increased deaths of endothelial cells in diabetes mellitus. However, the underlying mechanism is not fully understood. Because the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has a critical role in antagonizing PI3K pathways, we reasoned that peroxynitrite (ONOO–) generated by high blood sugar blocks PI3K/Akt activation by upregulating PTEN. In the present study, we demonstrate that ONOO– significantly increased the phosphorylation of LKB1, a tumor suppressor, resulting in enhanced association and phosphorylation of PTEN by LKB1, which increased the phosphorylation, stability, and activation of PTEN in both cultured endothelial cells and the aortas from diabetic mice. In addition, pharmacological or genetic inhibition of either PTEN or LKB1 prevents diabetes-enhanced apoptosis and Akt inhibition. Thus, our results suggest that diabetes mellitus, via hyperglycemia-driven ONOO–, resulted in accelerated apoptosis by LKB1-mediated but PTEN-dependent Akt inhibition. Our studies may lead to the development of therapeutic agents that protect against diabetes-related cardiovascular disease.


*    Footnotes
 
The online Data Supplement, consisting of an expanded Methods section, can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.716498/DC1.




This article has been cited by other articles: