FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2014125089> ?p ?o ?g. }

• W2014125089 endingPage "36344" @default.
• W2014125089 startingPage "36338" @default.
• W2014125089 abstract "Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis. Diabetes causes accelerated atherosclerosis and subsequent cardiovascular disease through mechanisms that are poorly understood. We have previously shown, using a porcine model of diabetes-accelerated atherosclerosis, that diabetes leads to an increased accumulation and proliferation of arterial smooth muscle cells in atherosclerotic lesions and that this is associated with elevated levels of plasma triglycerides. We therefore used the same model to investigate the mechanism whereby diabetes may stimulate smooth muscle cell proliferation. We show that lesions from diabetic pigs fed a cholesterol-rich diet contain abundant insulin-like growth factor-I (IGF-I), in contrast to lesions from non-diabetic pigs. Furthermore, two fatty acids common in triglycerides, oleate and linoleate, enhance the growth-promoting effects of IGF-I in smooth muscle cells isolated from these animals. These fatty acids accumulate predominantly in the membrane phospholipid pool; oleate accumulates preferentially in phosphatidylcholine and phosphatidylethanolamine, whereas linoleate is found mainly in phosphatidylethanolamine. The growth-promoting effects of oleate and linoleate depend on phospholipid hydrolysis by phospholipase D and subsequent generation of diacylglycerol. Thus, concurrent increases in levels of IGF-I and triglyceride-derived oleate and linoleate in lesions may contribute to accumulation and proliferation of smooth muscle cells and lesion progression in diabetes-accelerated atherosclerosis. smooth muscle cell arachidonic acid bovine serum albumin elaidic acid stearic acid diacylglycerol extracellular signal-regulated kinase high pressure liquid chromatography hydroxyoctadecadienoic acid insulin-like growth factor I lipoprotein lipase linoleic acid oleic acid phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphotidate phosphohydrolase phosphatidylinositol 3-kinase protein kinase B phospholipase A phospholipase C phospholipase D peroxisome proliferator-activated receptor phosphatidylserine p70 S6 kinase Both type 1 and type 2 diabetes result in an increased risk of developing atherosclerosis and subsequent myocardial infarction and stroke (1Ruderman N.B. Haudenschild C. Prog. Cardiovasc. Dis. 1984; 26: 373-412Crossref PubMed Scopus (251) Google Scholar). The mechanisms whereby diabetes accelerates atherosclerosis remain to be elucidated. A recently developed porcine model of diabetes-accelerated atherosclerosis demonstrates that the increased progression of atherosclerosis in diabetes is associated with increases in plasma triglycerides and blood glucose (2Gerrity R.G. Natarajan R. Nadler J.L. Kimsey T. Diabetes. 2001; 50: 1654-1665Crossref PubMed Scopus (216) Google Scholar). We have shown that in this model, diabetes leads to accumulation and proliferation of arterial smooth muscle cells (SMCs)1 in fibroatheromas and that high glucose levels are unable to directly increase proliferation of SMCs isolated from these pigs (3Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (174) Google Scholar). Thus, while diabetes increases the accumulation of SMCs in the atherosclerotic lesion, this appears to be independent of a direct growth-promoting effect of glucose. Our studies therefore focused on the association between elevated plasma triglycerides and lesion SMC proliferation in diabetes. There is strong evidence that higher plasma concentrations of fatty acids, a principal component of triglycerides, are correlated with increased risk of cardiovascular disease (4Cullen P. Am. J. Cardiol. 2000; 86: 943-949Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar) and are commonly present in humans and animals with diabetes (2Gerrity R.G. Natarajan R. Nadler J.L. Kimsey T. Diabetes. 2001; 50: 1654-1665Crossref PubMed Scopus (216) Google Scholar, 5Erkelens D.W. Eur. Heart. J. 1998; 19 Suppl. H: H27-H40PubMed Google Scholar). The major fatty acids in plasma triglycerides are palmitic acid (16:0), linoleic acid (cis18:2), stearic acid (18:0), oleic acid (cis 18:1), and arachidonic acid (cis 20:4). The results of this study show that IGF-I, a SMC growth factor (6Bayes-Genis A. Conover C.A. Schwartz R.S. Circ. Res. 2000; 86: 125-130Crossref PubMed Scopus (381) Google Scholar), is abundant in lesions of atherosclerosis from diabetic pigs, whereas lesions from non-diabetic pigs contain little IGF-I. Furthermore, oleic acid (OA) and linoleic acid (LA) markedly enhance the growth-promoting effects of IGF-I through a phospholipase D (PLD)-dependent pathway in SMCs isolated from these animals. We propose that the increased SMC proliferation seen in diabetes-accelerated atherosclerosis is, at least in part, caused by an increase in IGF-I-induced PLD activity and subsequent generation of diacylglycerol (DAG) fueled by the elevated levels of triglycerides containing OA and LA. Sodium salts of OA, LA, palmitic, arachidonic (AA), elaidic (EA), stearic acids (SA), and conjugated LA were purchased from Nucheck Prep (Elysian, MN). Human recombinant IGF-I was obtained from Upstate Biotechnology (Lake Placid, NY). [3H]Thymidine (6.7 Ci/mmol) was obtained from PerkinElmer Life Sciences. [14C]LA (55.0 mCi/mmol) and [14C]OA (56 mCi/mmol) were obtained from AmershamBiosciences. BSA, indomethacin, OA-CoA, LA-CoA, and a monoclonal anti-β-actin antibody were obtained from Sigma. Propranolol, atenolol, ONO-RS-082, U73312, U73343, R59022, L655238, baicalein, Wy-14643, LY294002, and wortmannin were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). 1-Butanol and 2-butanol were purchased from Fisher. All inhibitors were used at selective concentrations that did not result in cytotoxicity. Anti-phospho(S473)-PKB/AKT, anti-PKB/AKT, anti-phospho(T421/S424)-p70S6 kinase (p70S6K), anti-phospho-(T202/Y204)ERK antibodies, and anti-IGF-I receptor β-subunit antibodies were obtained from Cell Signaling Technologies (Beverly, MA). Anti-p70S6K antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbit polyclonal anti-ERK antibody (7884) generated against the extracellular signal-regulated kinase (ERK) subdomain-XI peptide RRITVEALAHPYLEQYYDPTDE was generously provided by Dr. Edwin G. Krebs. The porcine model of streptozotocin diabetes-accelerated atherosclerosis has been described in detail previously (2Gerrity R.G. Natarajan R. Nadler J.L. Kimsey T. Diabetes. 2001; 50: 1654-1665Crossref PubMed Scopus (216) Google Scholar, 3Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (174) Google Scholar). Fixed segments obtained from the area between the third and forth intercostal artery of the thoracic aorta were embedded in paraffin and cut into sections. The sections were prepared for immunohistochemistry as described previously (3Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (174) Google Scholar). Immunoreactive IGF-I was detected using a monoclonal anti-IGF-I antibody (Upstate Biotechnology) at a final concentration of 100 μg/ml. A control antibody of the same subclass (IgG1) and concentration was used as a negative control (Zymed Laboratories Inc.). IGF-I immunoreactivity was visualized by using an alkaline phosphatase kit (3Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (174) Google Scholar). SMCs were isolated from the thoracic aorta (between the third and forth intercostal artery) of non-diabetic pigs fed a chow diet via an explant method (3Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (174) Google Scholar). Passages 4–11 were used for experiments. Prior to all experiments, the SMCs were made quiescent by incubation in Dulbecco's modified Eagle's medium + 0.5% fetal bovine serum for 48 h. All experiments were performed in Dulbecco's modified Eagle's medium containing 5.6 mm glucose + 0.5% fatty acid-free-BSA. These conditions did not reduce cell viability or induce toxicity. Sodium salts of fatty acids were dissolved in distilled H2O (50 mg/ml) and diluted in sterile Dulbecco's modified Eagle's medium + 0.5% fatty acid-free-BSA (78 μm) to a final concentration of 70 μm. This mixture was equilibrated for 1 h at 37 °C in 5% CO2 prior to addition to the cells, allowing BSA–fatty acid complexes to form. This method has been estimated to result in an effective free fatty acid concentration in the nanomolar range (7Hamilton J.A. Kamp F. Diabetes. 1999; 48: 2255-2269Crossref PubMed Scopus (255) Google Scholar). The concentrations of fatty acid and BSA were based on a physiological ratio between fatty acid and the carrier protein (BSA) and the concentration of albumin present in the extracellular fluid of the intimal portion of the arterial wall (8Smith E.B. Staples E.M. Atherosclerosis. 1980; 37: 579-590Abstract Full Text PDF PubMed Scopus (125) Google Scholar). SMC proliferation was measured as [3H]thymidine incorporation into DNA and determination of cell number, as described previously (3Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (174) Google Scholar). Expression of the IGF-I receptor β-subunit and phosphorylation of downstream kinases were analyzed by immunoblotting. SMCs were stimulated with fatty acids and/or IGF-I for the indicated periods of time. Cell lysates were prepared as described previously (9Graves L.M. Bornfeldt K.E. Sidhu J.S. Argast G.M. Raines E.W. Ross R. Leslie C.C. Krebs E.G. J. Biol. Chem. 1996; 271: 505-511Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. After transfer, membranes were blocked in 5% nonfat milk in Tris-buffered saline-Tween 20 overnight at 4 °C. The membranes were then incubated overnight at 4 °C with primary antibodies according to the manufacturer's recommendations. Horseradish peroxidase-labeled secondary anti-rabbit and anti-mouse antibodies (Amersham Biosciences) were used at a 1:5000 dilution. Signal detection was performed by enhanced chemiluminescence (Amersham Biosciences). Analysis of fatty acid incorporation into membrane phospholipids was achieved via high pressure liquid chromatography (HPLC). SMCs in 100-mm dishes were labeled with [14C]LA or [14C]OA (3 μCi/plate) and incubated at 37 °C for 18 h. This procedure resulted in ∼90% incorporation of OA and LA into the cell layer. Membrane lipids were extracted using a modified Bligh and Dyer method (10Bligh E. Dyer W. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42389) Google Scholar), and lipid extracts were separated by normal-phase HPLC techniques (11Carroll M.A. Balazy M. Huang D.D Rybalova S. Falck J.R. McGiff J.C. Kidney Int. 1997; 51: 1696-1702Abstract Full Text PDF PubMed Scopus (95) Google Scholar). Reverse-phase HPLC was used to analyze the possible release of hydroperoxy lipids (11Carroll M.A. Balazy M. Huang D.D Rybalova S. Falck J.R. McGiff J.C. Kidney Int. 1997; 51: 1696-1702Abstract Full Text PDF PubMed Scopus (95) Google Scholar). The molecular species of endogenous cellular lipids were detected by UV monitoring at 206 nm and identified via comparison of elution times of authentic lipid standards. Radioactivity in the samples was measured using an online detector (β-RAM; Inus Systems, Tampa, FL). Analysis of OA and LA distribution into the neutral lipid pool was achieved via the use of TLC. Briefly, total lipids from SMCs incubated with 14C-labeled OA or LA were extracted as described above. Lipid extracts were applied to unmodified Silica Gel G TLC plates and developed in a solvent system of hexane:diethyl ether:glacial acetic acid (105:45:3) for ∼60 min. Plates were then dried and exposed to a phosphorimaging screen for 24 h. Standards containing mono-, di-, and triglycerides (Nucheck Prep) were visualized by exposure of the plate to iodine vapor. An Amersham Biosciences Storm 860 PhosphorImager was used for detection and quantification of radioactive spots. Results are expressed as mean ± S.E. of a minimum of three experiments, performed in triplicate. Data were analyzed by one-way analysis of variance, using Graph Pad Prism (Graph Pad Software, San Diego, CA). Simultaneous multiple comparisons were based on post-hoc comparison tests using a Student- Newman-Keuls test. Statistical significance was established at p< 0.05. IGF-I acts as a SMC growth factor and may play a role in lesion progression. We therefore determined levels of immunoreactive IGF-I in lesions from non-diabetic and diabetic pigs fed a cholesterol-rich diet. As shown in Fig.1 A, lesions from non-diabetic pigs fed a cholesterol-rich diet contained little IGF-I immunoreactivity. Lesions from diabetic pigs fed the same diet, on the other hand, were large and contained abundant IGF-I immunoreactivity that was localized mainly to the macrophage-rich region of the lesion (Fig. 1 B). A control antibody resulted in no staining of an adjacent section of the tissue (Fig. 1 C). Thus, lesions from diabetic pigs fed a cholesterol-rich diet contain more IGF-I immunoreactivity than lesions from non-diabetic animals. We evaluated the role of long-chained fatty acids common in triglycerides on basal and IGF-I-stimulated [3H]thymidine incorporation in porcine SMCs. We used a concentration of IGF-I (1 nm) that results in maximal stimulation of thymidine incorporation (data not shown). OA and LA potentiated the effects of IGF-I (Fig.2 A), whereas thetrans isomer of OA, EA, and the positional isomer of LA, conjugated LA (Fig. 2 A), did not potentiate the effects of IGF-I. Furthermore, other long-chained fatty acids, such as AA, SA, and palmitate, did not enhance the effects of IGF-I. 2B. Askari and K. E. Bornfeldt, unpublished observations. However, SA and palmitate exhibited cytotoxic effects at higher concentrations (>70 μm). To study the time course of OA- and LA-induced effects on IGF-I-stimulated thymidine incorporation, SMCs were pre-incubated for 0–24 h with 70 μm OA or LA and then stimulated with IGF-I for an additional 18 h. OA and LA potentiated the effects of IGF-I in a time-dependent manner, with a maximal effect observed after a 24-h preincubation with LA and a 6-h preincubation with OA (Fig. 2, B and C,hatched bars). Because fatty acids require the esterification to a CoA moiety catalyzed by acyl-CoA synthases to achieve biological activity (12Færgeman N.J. Knudsen J. Biochem. J. 1997; 323: 1-12Crossref PubMed Scopus (582) Google Scholar), we determined the effects of preincubation of the CoA esters of OA and LA on IGF-I-induced increases in thymidine incorporation. Fig. 2 (B and C) demonstrates that the effects of OA and LA are increased with their esterification to CoA, as both OA-CoA and LA-CoA were more effective in potentiating the mitogenic effects of IGF-I, and a significant potentiation was seen earlier than that of unmodified OA and LA (Fig.2, B and C, cross-hatched bars). To confirm these results, we analyzed the effects of OA and LA on IGF-I-mediated increases in SMC number. OA and LA significantly (p < 0.05) enhanced the effects of IGF-I after a 6-day stimulation (control: 54,970 ± 5,498 cells per well; LA, 61,830 ± 969 cells per well; OA, 68,843 ± 1,279 cells per well; IGF-I, 62,903 ± 786 cells per well; IGF-I + LA, 72,163 ± 3,307 cells per well; and IGF-I + OA, 81,270 ± 2,161 cells per well; mean ± S.E., n = 3). Thus, OA and LA enhance the growth-promoting effects of IGF-I in SMCs. We investigated the mechanism of action of OA- and LA-mediated enhancement of the IGF-I mitogenic effect. In some cell types, mitogenic effects of LA have been shown to be caused by the conversion of LA into its hydroxy metabolites, 9- and 13-HODEs, mostly via the action of the lipoxygenase pathway (13Rao G.N. Alexander R.W. Runge M.S. J. Clin. Invest. 1995; 96: 842-847Crossref PubMed Scopus (116) Google Scholar). However, our results show that LA-induced potentiation of the growth-promoting effects of IGF-I is independent of lipoxygenase activity, as pharmacological inhibition of either 5-lipoxygenase (L655238, 5 μm) or 12-lipoxygenase (baicalein, 25 μm) did not block the effect of LA (data not shown). It has also been demonstrated in other tissues that LA can serve as a substrate for cyclooxygenase (14Godessart N. Camacho M. Lopez-Belmonte J. Anton R. Garcia M. de Moragas J.M. Vila L. J. Invest. Dermatol. 1996; 107: 726-732Abstract Full Text PDF PubMed Scopus (28) Google Scholar). In the porcine SMCs, inhibition of cyclooxygenase-1/cyclooxygenase-2 with indomethacin (10 μm) had no effect (data not shown). Furthermore, HPLC analysis of the media of SMCs labeled with [14C]LA and stimulated with IGF-I confirmed the pharmacological data, as there was neither release into the media nor any esterification of 9-/13-HODEs into membrane phospholipids (data not shown). There is also evidence that conversion of LA into HODEs may lead to activation of peroxisome proliferator-activated receptors (PPARs) (15Willson T.M. Brown P.J Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1689) Google Scholar). However, it is unlikely that the effects of OA and LA on IGF-I-stimulated SMC proliferation are mediated by PPARs because other fatty acid PPAR activators, such as AA and EA did not mimic the effects. We also observed that the PPARα agonist Wy-14643 did not mimic the effects of OA and LA (data not shown). These results indicate that HODEs do not mediate the actions of LA on IGF-I-stimulated SMC proliferation. Mitogenic signaling pathways induced by the IGF-I receptor in SMCs were next studied. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been reported as a main mitogenic pathway activated by IGF-I in SMCs (16Imai Y. Clemmons D.R. Endocrinology. 1999; 140: 4228-4235Crossref PubMed Google Scholar), a finding that was confirmed in our studies (data not shown). As shown in Fig. 3, neither OA nor LA consistently enhanced IGF-I-induced phosphorylation or expression of either protein kinase B (PKB/AKT) or p70S6K, which act downstream of PI3K, or p42/p44 ERK for up to 24 h. Furthermore, expression of the IGF-I receptor β-subunit was not increased by OA or LA (Fig. 3). In mammalian cells, long-chain fatty acids undergo β-oxidation for use as an energy source, or they enter different lipid pools. However, neither OA nor LA undergoes significant β-oxidation in SMCs, as incubation of [14C]OA and [14C]LA did not lead to a detectable increase in release of 14CO2 for up to 24 h (data not shown). We therefore investigated the degree of OA and LA incorporation into different lipid pools in SMCs, using TLC. As shown in Table I, the majority (∼90%) of OA and LA is incorporated into the phospholipid pool, with minor amounts in triglycerides, diglycerides, and monoglycerides. We next determined which phospholipid pools were the destination of LA and OA, by using HPLC. Fig. 4, Aand B, demonstrates that following exposure of SMCs to exogenous OA and LA, LA is found mainly in membrane phosphatidylethanolamine (PE) and phosphatidylserine (PS) (Fig.4 A), whereas OA is distributed mainly into the PE, PS, and phosphatidylcholine (PC) pools (Fig. 4 B). These data demonstrate that the principal destination of OA and LA is the membrane phospholipid pools, which may serve as substrates for IGF-I-induced, phospholipase-dependent signaling pathways.Table IDistribution of OA and LA among membrane lipidsFAPLMG1,2DG1,3DGFFATGCEOA93 ± 1.30.03 ± 0.021.4 ± 0.40.12 ± 0.10.8 ± 0.44.5 ± 1.50.01 ± 0.01LA89 ± 3.10.02 ± 0.020.7 ± 0.30.07 ± 0.041.3 ± 0.68.6 ± 2.70.01 ± 0.01Porcine SMCs in 100-mm dishes were labeled with [14C]OA or [14C]LA (3 μCi/plate) for 24 h. Total lipids were extracted via a modified Bligh and Dyer method and run on TLC. This procedure separated the neutral lipids into phospholipids (PL), monoglycerides (MG), diglycerides (1,2DG and 1,3DG), free fatty acids (FFA), triglycerides (TG), and cholesterol esters (CE). Data are presented as mean ± S.E. of % of total incorporation (n = 4). Open table in a new tab Porcine SMCs in 100-mm dishes were labeled with [14C]OA or [14C]LA (3 μCi/plate) for 24 h. Total lipids were extracted via a modified Bligh and Dyer method and run on TLC. This procedure separated the neutral lipids into phospholipids (PL), monoglycerides (MG), diglycerides (1,2DG and 1,3DG), free fatty acids (FFA), triglycerides (TG), and cholesterol esters (CE). Data are presented as mean ± S.E. of % of total incorporation (n = 4). There are three groups of phospholipases that hydrolyze phospholipids, namely phospholipase C (PLC), phospholipase A2 (PLA2), and PLD. Becasue IGF-I may be able to activate all of these phospholipases, we investigated the contribution of PLC, PLA2, and PLD to OA and LA-induced potentiation of the mitogenic effect of IGF-I. Although IGF-I has previously been shown to activate PLC in SMCs (17Bornfeldt K.E. Raines E.W. Nakano T. Graves L.M. Krebs E.G. Ross R. J. Clin. Invest. 1994; 93: 1266-1274Crossref PubMed Scopus (380) Google Scholar), the results show that pharmacological inhibition of PLC (U73312, 3 μm) did not affect fatty acid-induced potentiation of IGF-I-stimulated DNA synthesis, indicating that PLC activity is not required for this effect (data not shown). The fact that neither OA nor LA is incorporated into phosphatidylinositol (Fig. 4) supports the lack of PLC involvement in the mitogenic effects induced by these fatty acids. Inhibition of PLA2 with arachidonyltrifluoromethyl ketone (AACOF3, 10 μm) or ONO-RS-082 (10 μm) also did not affect OA- and LA-mediated potentiation (data not shown). Furthermore, cytosolic PLA2 cleaves fatty acids off the sn-2 position of phosphatidylinositol (PI) and is associated with the production of LA- and AA-derived hydroperoxy metabolites (18Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). Our findings that OA and LA are not incorporated into phosphatidylinositol and that their effects are not mimicked by AA further support the lack of cytosolic PLA2 involvement, as does a previous study showing that IGF-I does not activate cytosolic PLA2 in SMCs (9Graves L.M. Bornfeldt K.E. Sidhu J.S. Argast G.M. Raines E.W. Ross R. Leslie C.C. Krebs E.G. J. Biol. Chem. 1996; 271: 505-511Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To determine the involvement of PLD, we used 1-butanol (an inhibitor of PLD action) and 2-butanol (inactive control). As shown in Fig.5 A, 1-butanol (0.2% v/v), but not 2-butanol (Fig. 5 B), completely blocked the potentiating effects of OA and LA. The role of the PLD pathway was also confirmed by using propranolol (a β-adrenergic blocker that also inhibits phosphotidate phosphohydrolase (PPH) activity). PPH catalyzes the conversion of phosphatidic acid (PA) to DAG. Atenolol, another β-blocker without PPH-blocking activity, was used as a negative control. Propranolol (Fig. 5 C), but not atenolol (Fig.5 D), blocked OA- and LA-induced potentiation of the growth-promoting effects of IGF-I. These results indicate that PLD activation mediates the effects of OA and LA. Because PLD cleaves membrane PC and PE to form PA, which in turn is metabolized into DAG via the activity of PPH, we hypothesized that elevation of DAG levels should mimic the effects of OA and LA. Therefore, we inhibited DAG kinase (which phosphorylates DAG to PA and thus decreases levels of DAG) by using R59022. Indeed, inhibition of DAG kinase dose-dependently increased IGF-I-induced thymidine incorporation (Fig. 6), thus mimicking the effects of OA and LA. Together, these results show that the effects of OA and LA depend on PLD activity and subsequent generation of DAG.Figure 6Inhibition of DAG kinase increases the proliferative effects of IGF-I in porcine SMCs. Porcine SMCs were pretreated with R59022 at the indicated concentrations for 30 min prior to the addition of IGF-I (1 nm). DNA synthesis was measured as described in Fig. 2. Values are represented as mean ± S.E. of triplicate samples of representative experiments (n = 3). *, p < 0.01 versus IGF-I alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We show that exposure of SMCs to physiological concentrations of OA and LA results in a marked enhancement of the growth-promoting effects of IGF-I, a growth factor present in lesions of atherosclerosis. Interestingly, OA and LA have previously been demonstrated to enhance the effects of other growth factors in SMCs, such as angiotensin II (19Lu G. Meier K.E. Jaffa A.A. Rosenzweig S.A. Egan B.M. Hypertension. 1998; 31: 978-985Crossref PubMed Scopus (47) Google Scholar, 20Lu G. Greene E.L. Nagai T. Egan B.M. Hypertension. 1998; 32: 1003-1010Crossref PubMed Scopus (69) Google Scholar, 21Greene E.L., Lu, G. Zhang D. Egan B.M. Hypertension. 2001; 37: 308-312Crossref PubMed Scopus (57) Google Scholar) and endothelin-1 (22Kwok C.F. Shih K.C. Hwu C.M. Ho L.T. Metabolism. 2000; 49: 1386-1389Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The results of the present study support and extend earlier findings (23Lu G. Morinelli T.A. Meier K.E. Rosenzweig S.A. Egan B.M. Circ. Res. 1996; 79: 611-618Crossref PubMed Google Scholar) that the ability to enhance growth factor effects appears to be specific to OA and LA since SA, palmitate, AA, the trans isomer of OA, and the positional isomer of LA (EA and conjugated LA, respectively), do not mimic the effects of OA and LA. It has also been shown that micromolar concentrations of OA and LA can induce proliferation of SMCs in the absence of growth factors when added without prior coupling to a carrier protein (13Rao G.N. Alexander R.W. Runge M.S. J. Clin. Invest. 1995; 96: 842-847Crossref PubMed Scopus (116) Google Scholar, 23Lu G. Morinelli T.A. Meier K.E. Rosenzweig S.A. Egan B.M. Circ. Res. 1996; 79: 611-618Crossref PubMed Google Scholar). Although our results indicate that OA and LA appear to exert weak mitogenic effects by themselves when complexed to BSA, their main effect is to enhance the growth-promoting effects of IGF-I. Thus, unbound OA and LA may induce cellular responses quite different from those observed when OA and LA are complexed to a carrier protein at a physiological ratio (7Hamilton J.A. Kamp F. Diabetes. 1999; 48: 2255-2269Crossref PubMed Scopus (255) Google Scholar). We show that the effects of OA and LA on IGF-I-mediated SMC proliferation are likely to depend on entry of OA and LA into the cell and subsequent generation of CoA-esterified fatty acids, since OA-CoA and LA-CoA induced potentiation of the IGF-I response more efficiently and rapidly than OA and LA. Indeed, ∼90% of incorporated OA and LA were found in the membrane phospholipid pool. However, it is important to note that there was a dissimilar distribution of OA and LA into the membrane pools because OA was distributed mainly into membrane PC, PE, and PS, whereas LA was distributed mainly into PE and PS. To investigate how OA- and LA-containing phospholipids enhance the mitogenic effects of IGF-I, we studied a number of different possibilities. Our results show that OA and LA do not result in increased expression of the IGF-I receptor β-subunit nor do they enhance the ability of IGF-I to phosphorylate downstream kinases implicated in the mitogenic effects of IGF-I. Furthermore, the effects of OA and LA are unlikely to be caused by increased formation of reactive oxygen species, corroborating a recent finding that the contribution of OA to mitochondrial oxidation is minor (24Allen T.J. Hardin C.D. J. Vasc. Res. 2001; 38: 276-287Crossref PubMed Scopus (7) Google Scholar). Accordingly, pretreatment with antioxidants had no effect on OA- or LA-induced potentiation of the growth-promoting effects of IGF-I. 3B. Askari and K. E. Bornfeldt, unpublished observations. Finally, under the conditions used in this study, neither activation of PPARs nor formation of HODEs appears to mediate the effects of OA and LA. Instead, our results show that the effects of OA and LA on IGF-I mitogenic action are mediated through a PLD-dependent mechanism. It has been shown, in SMCs as well as other cell types, that growth factors such as IGF-I can induce PLD activation (25Rydzewska G. Morisset J. Pancreas. 1995; 10: 59-65Crossref PubMed Scopus (25) Google Scholar, 26Exton J.H. Ann. N. Y. Acad. Sci. 2000; 905: 61-68Crossref PubMed Scopus (79) Google Scholar) and result in the hydrolysis of PC and, to some extent, PE. This hydrolysis leads to an increase in formation of PA, which is converted tosn-1, 2-DAG via the action of PPH (see Fig.7 and Ref. 26Exton J.H. Ann. N. Y. Acad. Sci. 2000; 905: 61-68Crossref PubMed Scopus (79) Google Scholar). Inhibition of either PLD or PPH resulted in attenuation of the OA- and LA-mediated potentiation of the growth-promoting effects of IGF-I. Thus, it is likely that increased DAG formation, not PA, mediates the effects of OA and LA. Furthermore, the fact that inhibition of DAG kinase mimics the effects of OA and LA further supports that PLD-mediated generation of DAG is responsible for our observations (Fig. 7). Therefore, we propose that the incorporation of OA and LA into membrane PC and PE, in combination with IGF-I-stimulated PLD activity, leads to an increase in OA- and LA-containing species of DAG that may have a higher degree of activity than the “native” DAG. Indeed, it has been reported that changing the molecular species of the acyl chains of DAG (27Marignani P.A. Epand R.M. Sebaldt R.J. Biochem. Biophys. Res. Commun. 1996; 225: 469-473Crossref PubMed Scopus (63) Google Scholar) can modulate the stimulation of protein kinase C activity, as measured in vitro. DAG has also been shown to activate other signaling molecules with potential mitogenic effects (28Wakelam M.J. Biochim. Biophys. Acta. 1998; 1436: 117-126Crossref PubMed Scopus (159) Google Scholar). Another possibility is that more DAG may be formed as a result of exposure of SMCs to OA and LA. Studies supporting this hypothesis have demonstrated that OA can inhibit platelet-derived growth factor–B-chain homodimer-induced increases in DAG kinase α activity in SMCs and that this attenuation of activity is associated with an increase in intracellular DAG concentrations (29Du X. Jiang Y. Qian W., Lu, X. Walsh J.P. Biochem. J. 2001; 357: 275-282Crossref PubMed Scopus (27) Google Scholar). We are currently investigating these alternate pathways. We have previously shown, using the porcine model of diabetes-accelerated atherosclerosis, that diabetes results in a marked increase in lesion progression and SMC accumulation and proliferation (3Suzuki L.A. Poot M. Gerrity R.G. Bornfeldt K.E. Diabetes. 2001; 50: 851-860Crossref PubMed Scopus (174) Google Scholar). The present study demonstrates a possible mechanism whereby elevated triglycerides and non-esterified fatty acids seen in diabetes may contribute to SMC proliferation and lesion progression. Accordingly, it has recently been shown that lipoprotein lipase (LPL), the main enzyme that hydrolyzes triglycerides into fatty acids, is expressed by macrophages in lesions of atherosclerosis (30O'Brien K.D. Gordon D. Deeb S. Ferguson M. Chait A. J. Clin. Invest. 1992; 89: 1544-1550Crossref PubMed Scopus (178) Google Scholar) and that overexpression of LPL can lead to an increase in the fatty acid content, preferentially OA, LA, and palmitic acid, in the arterial wall (31Esenabhalu V.E. Cerimagic M. Malli R. Osibow K. Levak-Frank S. Frieden M. Sattler W. Kostner G.M. Zechner R. Graier W.F. Br. J. Pharm. 2002; 135: 143-154Crossref PubMed Scopus (27) Google Scholar). Furthermore, when macrophages are deficient in LPL, there is a decreased formation/progression of lesions of atherosclerosis (32Babaev V.R. Patel M.B. Semenkovich C.F. Fazio S. Linton M.F. J. Biol. Chem. 2000; 275: 26293-26299Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 33Clee S.M. Bissada N. Miao F. Miao L. Marais A.D. Henderson H.E. Steures P. McManus J. McManus B. LeBoeuf R.C. Kastelein J.J. Hayden M.R. J. Lipid Res. 2000; 41: 521-531Abstract Full Text Full Text PDF PubMed Google Scholar, 34Van Eck M. Zimmermann R. Groot P.H. Zechner R. Van Berkel T.J. Arterioscler. Thromb. Vasc. Biol. 2000; 20: e53-e62Crossref PubMed Google Scholar). Our preliminary results show that levels of immunoreactive LPL are markedly elevated in the macrophage-rich area in lesions from diabetic pigs compared with non-diabetic pigs. 4B. Askari, F. Kramer, and K. E. Bornfeldt, unpublished observations. Interestingly, high glucose levels and advanced glycation end products have been shown to increase LPL (35Sartippour M.R. Lambert A. Laframboise M., St- Jacques P. Renier G. Diabetes. 1998; 47: 431-438Crossref PubMed Scopus (33) Google Scholar) expression in macrophages. In the present study, lesions from diabetic pigs were found to contain more IGF-I immunoreactivity than lesions from non-diabetic pigs. The IGF-I associated mainly with macrophage-rich areas of the lesions. We are currently investigating the possible mechanisms responsible for this increased IGF-I immunoreactivity. One possibility is that macrophages are the main source of IGF-I in the lesion and that lesions from diabetic pigs contain more macrophages than lesions from non-diabetic pigs. Other possibilities include a direct effect of factors associated with the diabetic environment on macrophage expression of IGF-I (36Kirstein M. Aston C. Hintz R. Vlassara H. J. Clin. Invest. 1992; 90: 439-446Crossref PubMed Scopus (237) Google Scholar) or trapping of IGF-I derived from circulation, or other lesion cell types, in macrophage-rich areas. Whatever the source of IGF-I (endocrine, paracrine, or autocrine), the growth-promoting effects of IGF-I on SMCs are enhanced by OA and LA. Thus, we propose that diabetes results in increased levels of LPL and IGF-I in atheromas and that these events, in combination with the elevated levels of OA- and LA-containing triglycerides, accelerate SMC proliferation and lesion progression." @default.
• W2014125089 created "2016-06-24" @default.
• W2014125089 creator A5011658040 @default.
• W2014125089 creator A5017080599 @default.
• W2014125089 creator A5045860181 @default.
• W2014125089 creator A5056975442 @default.
• W2014125089 creator A5062756168 @default.
• W2014125089 creator A5069775294 @default.
• W2014125089 date "2002-09-01" @default.
• W2014125089 modified "2023-09-29" @default.
• W2014125089 title "Oleate and Linoleate Enhance the Growth-promoting Effects of Insulin-like Growth Factor-I through a Phospholipase D-dependent Pathway in Arterial Smooth Muscle Cells" @default.
• W2014125089 cites W1763805809 @default.
• W2014125089 cites W1967535453 @default.
• W2014125089 cites W1977666051 @default.
• W2014125089 cites W1987945927 @default.
• W2014125089 cites W1988129870 @default.
• W2014125089 cites W2008658973 @default.
• W2014125089 cites W2017038002 @default.
• W2014125089 cites W2022996249 @default.
• W2014125089 cites W2029447015 @default.
• W2014125089 cites W2044918250 @default.
• W2014125089 cites W2051870109 @default.
• W2014125089 cites W2065669726 @default.
• W2014125089 cites W2071115174 @default.
• W2014125089 cites W2082558448 @default.
• W2014125089 cites W2083531486 @default.
• W2014125089 cites W2087525053 @default.
• W2014125089 cites W2088927230 @default.
• W2014125089 cites W2094856121 @default.
• W2014125089 cites W2107327751 @default.
• W2014125089 cites W2107391327 @default.
• W2014125089 cites W2109836982 @default.
• W2014125089 cites W2118678876 @default.
• W2014125089 cites W2123276507 @default.
• W2014125089 cites W2126568311 @default.
• W2014125089 cites W2134170175 @default.
• W2014125089 cites W2139933399 @default.
• W2014125089 cites W2152311269 @default.
• W2014125089 cites W2152859407 @default.
• W2014125089 cites W2154941544 @default.
• W2014125089 cites W2155278362 @default.
• W2014125089 cites W2160677996 @default.
• W2014125089 cites W2164221407 @default.
• W2014125089 cites W4248088850 @default.
• W2014125089 cites W4248395277 @default.
• W2014125089 doi "https://doi.org/10.1074/jbc.m205112200" @default.
• W2014125089 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12138107" @default.
• W2014125089 hasPublicationYear "2002" @default.
• W2014125089 type Work @default.
• W2014125089 sameAs 2014125089 @default.
• W2014125089 citedByCount "40" @default.
• W2014125089 countsByYear W20141250892012 @default.
• W2014125089 countsByYear W20141250892013 @default.
• W2014125089 countsByYear W20141250892014 @default.
• W2014125089 countsByYear W20141250892016 @default.
• W2014125089 countsByYear W20141250892018 @default.
• W2014125089 countsByYear W20141250892021 @default.
• W2014125089 countsByYear W20141250892022 @default.
• W2014125089 crossrefType "journal-article" @default.
• W2014125089 hasAuthorship W2014125089A5011658040 @default.
• W2014125089 hasAuthorship W2014125089A5017080599 @default.
• W2014125089 hasAuthorship W2014125089A5045860181 @default.
• W2014125089 hasAuthorship W2014125089A5056975442 @default.
• W2014125089 hasAuthorship W2014125089A5062756168 @default.
• W2014125089 hasAuthorship W2014125089A5069775294 @default.
• W2014125089 hasBestOaLocation W20141250891 @default.
• W2014125089 hasConcept C126322002 @default.
• W2014125089 hasConcept C12927208 @default.
• W2014125089 hasConcept C134018914 @default.
• W2014125089 hasConcept C170493617 @default.
• W2014125089 hasConcept C181199279 @default.
• W2014125089 hasConcept C185592680 @default.
• W2014125089 hasConcept C2775960820 @default.
• W2014125089 hasConcept C2776551241 @default.
• W2014125089 hasConcept C2779306644 @default.
• W2014125089 hasConcept C2992686903 @default.
• W2014125089 hasConcept C55493867 @default.
• W2014125089 hasConcept C6182249 @default.
• W2014125089 hasConcept C62478195 @default.
• W2014125089 hasConcept C71924100 @default.
• W2014125089 hasConcept C86803240 @default.
• W2014125089 hasConcept C95444343 @default.
• W2014125089 hasConceptScore W2014125089C126322002 @default.
• W2014125089 hasConceptScore W2014125089C12927208 @default.
• W2014125089 hasConceptScore W2014125089C134018914 @default.
• W2014125089 hasConceptScore W2014125089C170493617 @default.
• W2014125089 hasConceptScore W2014125089C181199279 @default.
• W2014125089 hasConceptScore W2014125089C185592680 @default.
• W2014125089 hasConceptScore W2014125089C2775960820 @default.
• W2014125089 hasConceptScore W2014125089C2776551241 @default.
• W2014125089 hasConceptScore W2014125089C2779306644 @default.
• W2014125089 hasConceptScore W2014125089C2992686903 @default.
• W2014125089 hasConceptScore W2014125089C55493867 @default.
• W2014125089 hasConceptScore W2014125089C6182249 @default.
• W2014125089 hasConceptScore W2014125089C62478195 @default.
• W2014125089 hasConceptScore W2014125089C71924100 @default.
• W2014125089 hasConceptScore W2014125089C86803240 @default.
• W2014125089 hasConceptScore W2014125089C95444343 @default.

• next