Reduced Expression of Adipose Triglyceride Lipase Enhances Tumor Necrosis Factor α-induced Intercellular Adhesion Molecule-1 Expression in Human Aortic Endothelial Cells via Protein Kinase C-dependent Activation of Nuclear Factor-κB*

Background: Adipose triglyceride lipase (ATGL) expression is decreased in the obese insulin-resistant state. Results: RNA interference-mediated down-regulation of ATGL enhanced monocyte adhesion via increased expression of tumor necrosis factor α-induced intercellular adhesion molecule-1. Conclusion: Reduced ATGL expression may influence the atherogenic process in the insulin-resistant state. Significance: This mechanism also leads to the acceleration of atherosclerosis in patients with insulin-resistance, even if not in a state of hyperglycemia. We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.

We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNF␣-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IB␣ were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNF␣-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNF␣-induced ICAM-1 expression via acti-vation of NFB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.
Heart diseases, including coronary heart disease, cardiomyopathy, and heart failure, are the major causes of early mortality in individuals with diabetes mellitus. Despite recent progress in medical, interventional, and surgical treatments, the prognoses for these heart diseases are still worse in diabetics than in nondiabetics; thus, it is important to further elucidate the pathophysiology and mechanisms underlying diabetes-related heart diseases and to identify a novel therapeutic target.
Myocardial triglyceride (TG) 2 content is significantly higher in patients with prediabetes or diabetes than in healthy volunteers (1,2) and is associated with impaired left ventricular diastolic function (1). However, the role played by TG accumulation in atherosclerosis, including that of the coronary arteries, remains unknown.
Neutral lipid storage disease with myopathy is characterized by the presence of intracellular TG deposition in most tissues due to mutations in adipose triglyceride lipase (ATGL) (3), which catalyzes the first step in the hydrolysis of TG stored within lipid droplets (4). Triglyceride deposit cardiomyovasculopathy, a cardiac phenotype of neutral lipid storage disease with myopathy, shows TG accumulation in the coronary arteries and the myocardium, leading to concentric and diffuse coronary atherosclerotic lesions and chronic heart failure, both common in diabetics. Because the expression and enzymatic * This work was supported by research grants-in-aid for rare and intractable diseases from the Ministry of Health, Labor, and Welfare of the Japanese Government (to K. H. and K. K.). 1 To whom correspondence should be addressed: activity of the ATGL protein are reduced in obese subjects and in the insulin-resistant state (5,6), ATGL may be related to the high incidence of accelerated atherosclerosis and cardiomyopathy seen in individuals with diabetes mellitus. Several adhesion molecules are expressed on endothelial cells and play an important role in monocyte-endothelium interactions during the initiation and progression of atherosclerosis. intercellular adhesion molecule-1 (ICAM-1), a member of the immunoglobulin supergene family, is involved in immune and inflammatory responses (7) because it is critical for mediating monocyte adhesion to the endothelium (8) and the transmigration of leukocytes. A previous study (9) reported elevated levels of ICAM-1 in diabetes. Also, nuclear transcription factor-B (NFB) is an important factor involved in the transcriptional regulation of ICAM-1 stimulated by tumor necrosis factor ␣ (TNF␣) (10). In this report, we investigated how ATGL knockdown affected monocyte adhesion to the endothelium and TNF␣-induced ICAM-1 expression and NFB activation in human aortic endothelial cells (HAECs).
Cells-HAECs were obtained from Cambrex (Walkersville, MD) and grown in 75-cm 2 culture flasks (250,000 cells/flask) (Corning Glass) at 37°C in 5% CO 2 in endothelial basal medium-2 supplemented with endothelial cell growth medium SingleQuots (Cambrex). HAECs were used at passages five or six. Human monocytic U937 cells were purchased from the Human Sciences Research Resource Bank (Tokyo, Japan) and grown in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (Hyclone, Victoria, Australia), 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .
Small Interfering RNA Transfection-HAECs were plated at a density that achieved 50 -80% confluence on the day of transfection. Lipofectamine RNAiMAX (Invitrogen) and ATGL Stealth siRNA or a Stealth siRNA-negative universal control (Invitrogen) were first incubated in Opti-MEM I reduced serum medium (Invitrogen) and added to the HAECs in antibiotic-free medium. Nucleotides 575-599 (GenBank TM accession number NM020376) of human ATGL cDNA (5Ј-GAGAATGTCATTATATCCCACTTCA-3Ј) were used as the target sequence for RNAi-mediated knockdown.
Monocyte Adhesion Assay-HAECs were grown in 6-well plates and transfected with control siRNA or ATGL siRNA. On the day of the experiment, the medium was removed from each well, the cells were washed with PBS, and fresh medium containing U937 cells (3 ϫ 10 5 cells/well) was added to each well and incubated for 15 min at room temperature on a rocking plate. After washing with PBS, adherent U937 cells were fixed in 1% paraformaldehyde. The number of adherent cells was counted in five different fields using an ocular grid (0.01 mm 2 /field).
The linearity of the amplifications as a function of cycle number was tested in preliminary experiments, and the mRNA expression levels were normalized to the expression levels of the housekeeping gene ␤-actin.
Measurement of Intracellular Diglycerides-Cells were harvested and washed twice with 150 mM NaCl, and total lipids were extracted using the Folch partition method (11). Briefly, cells were dissolved in 7.5 ml of chloroform/methanol/water (8:4:3, v/v/v) and allowed to stand for 30 min at room temperature. After centrifugation at 500 ϫ g for 10 min, the lower phase was collected. The upper phase was mixed with 4 ml of chloroform, and diacylglycerol (DAG) was re-extracted as outlined above. The lower phases (containing lipid) from the two centrifugation steps were combined and dried under nitrogen gas. Total diacylglycerol content and that of the various molecular species of diacylglycerol were measured using high performance liquid chromatography-tandem mass spectrometry, as described previously (12).
Labeling of DAG-HAECs were incubated with medium containing 2% fetal bovine serum (FBS) and [ 3 H]palmitate (20 Ci/ ml). The reaction was terminated, and total lipids were extracted as described above. First, the radioactivity of the total cell lipid extract was measured to evaluate the effects of ATGL knockdown on the incorporation of labeled fatty acids into the cells. Next, labeled DAG was separated on silica gel G thin layer plates developed in hexane/ether/acetic acid (60:40:1). The DAG spots (visualized using iodine gas) were scraped, and the radioactivity of the samples was determined by liquid scintillation counting (13).
Statistical Analysis-All data were expressed as the mean Ϯ S.E. Statistical analysis was performed using Student's t test, and p Ͻ 0.05 was considered statistically significant.

ATGL Knockdown Increases TNF␣-induced ICAM-1 Ex-
pression-Adhesion of circulating monocytes to the endothelium is one of the key events during the early stages of atherosclerogenesis (14). Because ICAM-1 mediates monocyte adhesion to the endothelium, we examined the expression of TNF␣-induced ICAM-1 in ATGL knockdown HAECs. As shown in Fig. 1A, 24 -72 h after siRNA transfection, we observed a Ͼ95% decrease in ATGL mRNA expression in  ATGL knockdown cells compared with that in cells transfected with the control. ATGL protein expression also decreased in ATGL knockdown HAECs 24 h after transfection (Fig. 1B). We next found that both the mRNA and protein levels of TNF␣induced ICAM-1 increased in HAECs treated with ATGL siRNA (Fig. 2, A and B). ATGL knockdown alone had no significant effect on ICAM-1 levels (Fig. 2C). Next, we examined whether ATGL knockdown affected TNF␣-stimulated adhesion of human monocytoid U937 cells to HAECs. As shown in Fig. 3, treatment of HAECs with TNF␣ (1 ng/ml) for 18 h resulted in a marked increase in the adhesion of U937 cells. ATGL knockdown alone had no significant effect on U937 cell adhesion, but it significantly increased the number of U937 cells adhering to HAECs in the presence of TNF␣.
The Signal Transduction Pathway Leading to Synthesis of Adhesion Molecules Involves Activation of NFB-NFB activation by inflammatory cytokines (including TNF␣) results in the induction of various endothelial cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and ICAM-1. These molecules participate in the recruitment of leukocytes to inflammatory lesions (10,15). We next determined the effect of ATGL knockdown on the degradation of IB␣ and the expression of NFB in HAECs and found that the amount of IB␣ protein was markedly decreased 15 min after TNF␣ stimulation (Fig. 4A). Pretreatment with ATGL siRNA significantly increased TNF␣-mediated degradation of IB␣ at 15 and 30 min but did not affect the expression of NFB (Fig. 4B).
Effect of ATGL Knockdown on Diacylglycerol Metabolism-Free fatty acids activate PKC via increased synthesis of DAG, a potent activator of PKC (19,20). We next examined the effect of ATGL knockdown on DAG levels and found that total DAG levels were increased 1.3-fold in ATGL knockdown HAECs (Fig. 7). We also observed significantly higher levels of 1-palmitoyl-2-oleoylglycerol (C16:0 -18:1) and 1,2-dioleoylglycerol (C16:0 -16:0) (Fig. 7). Next, labeling studies were used to further examine the mechanisms underlying the increase in DAG. ATGL knockdown increased the incorporation of [ 3 H]palmitate into HAECs (Fig. 8A). Furthermore, the level of labeled DAG in ATGL knockdown HAECs significantly increased when the cells were incubated with [ 3 H]palmitate (Fig. 8B). Because transmembrane transport of free fatty acids is actively facilitated by transporter proteins, we examined the gene expression of the fatty acid transporter, CD36. CD36 expression increased dramatically in ATGL knockdown HAECs (Fig.  9, A and B). PPAR␥ is essential for the basal regulation of CD36, and many factors regulate CD36 expression through a PPAR␥dependent mechanism (21). We found that ATGL knockdown increased PPAR␥ mRNA expression in HAECs (Fig. 10). Taken together, these data demonstrate that DAG synthesis increases via increased CD36-mediated fatty acid transportation in ATGL knockdown HAECs.
The classical enzyme, HSL, hydrolyzes TG, DAG, and monoacylglyceride but has highest specific activity against DAG (22). We next determined the effect of ATGL knockdown on HSL activation. The expression levels of HSL mRNA and HSL phosphoprotein were unchanged in ATGL knockdown HAECs (Fig.  11, A and B). This suggests that increased DAG synthesis, along with an impaired compensatory increase in HSL-mediated DAG hydrolysis, may contribute to increased DAG levels in ATGL knockdown HAECs.

DISCUSSION
Heart diseases, including ischemic heart disease and heart failure, are the major cause of mortality in diabetics, despite recent developments in medical and surgical treatment. Therefore, it is important to elucidate the underlying mechanisms and to identify new therapeutic targets. Increased TG levels in plasma and tissues are a common feature of the diabetic state. This abnormal TG metabolism may contribute to heart diseases in diabetics. Lingvay et al. (23) reported that increased TG content in the myocardium, a common feature in diabetics, is associated with ventricular diastolic dysfunction. However, it remains unclear whether TG deposition plays a role in the process of atherosclerosis. To clarify this, we focused our attention on ATGL, a recently discovered TG lipase (4). ATGL is the causative gene of triglyceride deposit cardiomyovasculopathy, which is characterized by massive accumulation of TG in the coronary arteries and myocardium and leads to chronic heart failure (24). Additionally, in patients with prediabetes or diabetes, ATGL protein expression and TG lipase activity are both reduced (5,6). Therefore, we investigated whether decreased ATGL activity may affect several biomolecules involved in the process of atherosclerosis in vitro.
Endothelial cell activation by various inflammatory stimuli, including TNF␣, increases the adherence of monocytes, a crucial step in the development of vascular diseases (15). We assessed the effect of ATGL activity on U937 adhesion to HAECs stimulated with TNF␣. Our results show that ATGL knockdown in HAECs stimulated with TNF␣ up-regulates the expression of the adhesion molecule, ICAM-1, resulting in enhanced monocyte adhesion. This finding indicates that ATGL acts as an endogenous modulator of endothelial inflammatory responses and the adherence of monocytes.
NFB regulates the expression of numerous proteins involved in inflammation, including cytokines (IL-6), chemokines (IL-8, MCP-1, and regulated upon activation, normal T  cell expressed and secreted (RANTES)), and cell adhesion molecules (ICAM-1 and VCAM-1) (25). The NFB transcription factor is present in the cytosol in an inactive state complexed with the inhibitory protein, IB (26). Activation occurs via phosphorylation of IB␣ at Ser-32 and Ser-36, followed by proteasome-mediated degradation, resulting in the release and nuclear translocation of active NFB (27). In the present study, we showed that the amount of IB␣ protein in ATGL knockdown HAECs was markedly decreased 15 min after TNF␣ stimulation. These results suggest that ATGL knockdown increases ICAM-1 expression via activation of NFB. The signaling pathways mediating these effects involve p42/44 MAPK, p38 MAPK, and PKC (28,29). We showed that phosphorylation of PKC was increased in ATGL knockdown HAECs and that calphostin C and GF109203X inhibit TNF␣-induced ICAM-1 expression. We concluded, therefore, that ATGL knockdowninduced expression of ICAM-1 was mediated by PKC-dependent NFB activation.
PKCs are activated by DAG, phosphatidylserine, and phorbol esters. Although the absence of ATGL would be expected to   reduce both TG hydrolysis and other lipid metabolites, DAG concentrations were, surprisingly, increased in ATGL knockout mice (30). We found that PKC was already activated before TNF␣ stimulation in ATGL knockdown HAECs (Fig. 5D). Therefore, we speculated that increased DAG levels induced by ATGL knockdown activate PKC and found that DAG levels were increased in ATGL knockdown HAECs (Fig. 7). Interestingly, the fatty acid composition of DAG derived from de novo synthesis is supposed to be rich in palmitate and oleate, whereas DAG derived from phosphatidylinositol breakdown is rich in arachidonate (13,31). In the present study, we found that the levels of diacylglycerol species containing palmitic acid (C16:0) and oleic acid (C18:1) were increased in ATGL knockdown HAECs, but the levels of diacylglycerol species containing arachidonic acid (C20:4) showed no significant differences (Fig.  7). These findings suggested that DAGs derived from de novo synthesis were increased in ATGL knockdown HAECs. We previously reported that saturated non-esterified fatty acids can also stimulate de novo DAG synthesis and PKC activity in cultured aortic endothelial cells (32) and vascular smooth muscle cells (19). In the present study, we showed that fatty acid uptake, DAG synthesis, and the expression of CD36 (a major transporter for oxidized low density lipoprotein and long chain fatty acids) were increased in ATGL knockdown HAECs. This suggests that increased DAG synthesis due to uptake of fatty acids via up-regulated CD36 causes PKC activation in ATGL knockdown HAECs. However, we did not determine which isoform of PKC is involved in the regulation. It was reported that enhanced fatty acid flux increases the activation of the PKC isoforms, PKC␣/␤II, and of NFB (20). Therefore, PKC␣/␤II might be involved in PKC activation induced by ATGL knockdown; however, further studies are necessary. Because ATGL hydrolyzes TG, DAG, and monoacylglyceride, the absence of ATGL-mediated DAG hydrolysis, with the absence of a compensatory increase in HSL, may also contribute to increased DAG levels. Additionally, enhanced CD36 expression stimulates the uptake of oxidized low density lipoprotein, leading to the excessive acceleration of atherosclerosis in the presence of low ATGL activity, as seen in insulin resistance and diabetes.
In recent years, oxidative stress has been considered an important pathogenic factor in the development of diabetic vascular complications (33)(34)(35). Accumulating evidence shows that many protein, lipid, and DNA markers of oxidative stress are increased in the kidney and vascular tissues from animals and patients with diabetes (35)(36)(37). We previously reported that high glucose levels stimulate superoxide production via the PKC-dependent activation of NAD(P)H oxidases in cultured aortic endothelial cells and smooth muscle cells (38). The mechanisms underlying PKC-dependent activation of NAD(P)H oxidase were thought to act via PKC-dependent activation of the small GTPase, Rac-1, which is an important regulator of NAD(P)H oxidase activation (39). Activated PKC phosphorylates p47 phox and induces the release of superoxide from NOX components. In the present study, we found that both phosphorylation of PKC and DAG synthesis were increased in ATGL knockdown HAECs, suggesting that reduced ATGL expression may increase oxidative stress mediated by activated PKC without high glucose or increased free fatty acid. This mechanism also leads to the acceleration of atherosclerosis in triglyceride deposit cardiomyovasculopathy and in patients with insulin resistance, even if not in a state of hyperglycemia or increased free fatty acids.
PPAR␥ is essential for the basal regulation of CD36, and many factors regulate CD36 expression through a PPAR␥-dependent mechanism (21). In agreement with another report (21), we found that that PPAR␥ mRNA expression was increased in ATGL knockdown HAECs.
In conclusion, our results show for the first time that PKC is activated by increased fatty acid uptake and DAG synthesis in ATGL knockdown HAECs, at least in part, via increased expression of CD36. Activation of PKC leads to NFB activation and, in turn, to increased expression of TNF␣-induced ICAM-1 genes. In humans, there are many secretory products from adipocytes, including free fatty acids and TNF␣, which are known to modulate multiple physiological functions. In the obese insulin-resistant state, decreased ATGL activity, coupled with increased TNF␣ levels, is likely to contribute to the chronic inflammatory events associated with atherosclerosis.