The occurrence of coronary heart disease (CHD) and other manifestations of atherosclerotic vascular disease are substantially increased in patients with type 2 diabetes. Mortality from CHD and the incidence of nonfatal CHD events are two to four times higher in patients with type 2 diabetes than in age-matched nondiabetic subjects (1,2). It has been demonstrated that lipoprotein lipase (LPL), a secretory product of macrophage in the arterial wall, contributes to the development and progression of atherosclerosis (3). Michaud et al. (4) recently demonstrated that fatty acids enhance LPL production in human macrophages. Type 2 diabetic patients frequently have higher serum nonesterified fatty acids (NEFAs) (5). From these reports, it may be suggested that fatty acids participate in the development and progression of atherosclerosis in type 2 diabetic patients. To the best of our knowledge, however, the relationship between serum fatty acids and the degree of atherosclerosis has not been fully clarified in type 2 diabetic patients.

Plasma glucose level per se seems to enhance LPL production in human macrophage (6). Moreover, it is well recognized that obesity and/or hypertension per se causes atherosclerosis in type 2 diabetic patients. We therefore recruited nonobese nonhypertensive well-controlled unique type 2 diabetic patients after taking into account these confounding risk factors. The degree of atherosclerosis can be evaluated by high-resolution B-mode ultrasound scan. This is a reliable noninvasive method for the assessment of carotid atherosclerosis (7). Carotid atherosclerosis is important in view of its relation to cerebrovascular ischemic diseases and coronary atherosclerosis (8).

A total of 54 nonobese nonhypertensive Japanese type 2 diabetic patients who visited Kansai-Denryoku Hospital were enrolled in the study. Type 2 diabetes and hypertension were diagnosed based on the criteria of World Health Organization (9,10). The patients were treated with diet alone (27 patients) or diet in combination with sulfonylurea (27 patients). No patients were treated with insulin or antihypertensive medications. All subjects had ingested at least 150 g of carbohydrate for the 3 days preceding the study. None of the subjects had significant renal, hepatic, or cardiovascular disease. They did not receive any medications affecting lipid metabolism. They did not consume alcohol or perform heavy exercise for ≥1 week before the study.

Blood was drawn in the morning after a 12-h fast. Plasma glucose was measured with the glucose oxidase method and serum insulin was measured using a two-site immunoradiometric assay (Insulin Riabead II; Dainabot, Osaka City, Japan). Coefficients of variation (CVs) were 4% for insulin >25 μU/ml and 7% for insulin <25 μU/ml, respectively. The triglycerides, total cholesterol, and HDL cholesterol were also measured. LDL cholesterol was calculated using the Friedewald formula (11). Serum NEFAs were measured in duplicate using enzymatic method (NEFA HR kit; Wako Chemicals, Osaka, Japan), and the mean of the two values was used (12). The CV for NEFA was 2%. Blood pressure was measured twice in the sitting position, and the average was taken.

A carotid sonography was performed with high-resolution B-mode scanning equipment (Logic 400 GE; GE Yokogawa, Milwaukee, WI) with a 7.5-MHz sector scanner probe. The common carotid arteries of both sides were examined with longitudinal and transverse scans, because we could not fully analyze the internal and external carotid arteries in all patients. The CV for interobserver variability was found to be 8.5% and the CV for intraobserver variability was 6.0%. The intimal plus medial thickness (IMT) of the common carotid artery was measured in plaque-free segments as the distance from the leading edge of the first echogenic line corresponding to the lumen-intimal interface to the second echogenic line corresponding to the collagen-contained upper layer of tunic adventitia (13). The mean of IMT in plaque-free segments of bilateral common carotid arteries was used for the analysis. The degree of stenosis was also measured in the plaque segments of bilateral common carotid arteries. It was calculated as a percentage ratio between the area of the plaque and that of the lumen using the formula (lumen area − residual lumen) × 100 (14). Both of the areas were automatically measured by the system on a frozen transverse scanning plane at the site of maximal narrowing. When two or more plaques were present in the vessel, only the one causing the greatest degree of stenosis was considered for analysis.

The statistical analyses were conducted using the StatView 5 system (Statview, Berkeley, CA). Simple (Spearman’s rank) correlation coefficients between the degree of carotid atherosclerosis (IMT and carotid stenosis) and measures of variables were calculated, and a stepwise multiple regression analysis was then used to evaluate the independent association of these variables with the degree of carotid atherosclerosis. Data were presented as means ± SEM unless otherwise stated. P < 0.05 was considered significant. In multivariate analysis, F ≥ 4 was considered significant.

The subjects studied were 54 nonhypertensive Japanese type 2 diabetic patients (41 men and 13 women) with an age of 59.8 ± 1.4 years and a BMI of 22.6 ± 0.3 kg/m2. They all were nonobese (BMI <27.0 kg/m2) (15). The duration of diabetes was 9.5 ± 1.0 years. Systolic and diastolic blood pressure was 124 ± 2 mmHg (range 92–155) and 72 ± 1 (58–90), respectively. Fasting plasma glucose was 153 ± 5 mg/dl and HbA1c was 7.0 ± 0.2%. Fasting insulin level was 6.4 ± 0.4 μU/ml. Serum triglycerides, total cholesterol, and HDL cholesterol levels were 119 ± 8, 190 ± 4, and 51 ± 2 mg/dl, respectively. LDL cholesterol level was 115 ± 3 mg/dl. Serum NEFA level was 0.61 ± 0.03 mEq/l. Mean IMT in plaque-free segments and the degree of carotid stenosis (% stenosis) was 0.71 ± 0.02 mm and 8.1 ± 2.1%, respectively.

Spearman’s rank correlations of mean IMT in plaque-free segments or the degree of carotid stenosis with measures of variables were calculated for all of our diabetic patients. IMT in plaque-free segments was positively correlated with age (r = 0.502, P = 0.0003) and NEFA (r = 0.378, P = 0.0096). The degree of stenosis was positively correlated to age (r = 0.431, P = 0.0017), duration of diabetes (r = 0.307, P = 0.0255), and NEFA (r = 0.544, P = 0.0001).

Next, multiple regression analyses were carried out using the stepwise procedure. The analysis included IMT or the degree of stenosis as a dependent variable and candidate risk factors as independent variables. IMT in plaque-free segments was independently predicted by age (F = 16.5), which explained 24.8% of the variability of IMT in our diabetic patients. In contrast, the degree of stenosis was independently associated with NEFA (F = 10.5), which explained 16.8% of the variability of the carotid stenosis in our type 2 diabetic patients. Other variables, including BMI and lipid profile, were not associated with either IMT in plaque-free segments or the degree of carotid stenosis in our patients.

It is generally accepted that atherosclerosis and related vascular disorders are the leading cause of death in type 2 diabetic patients. Several factors are associated with atherosclerosis in diabetes. Bierman (16) previously estimated that typical risk factors, including smoking, cholesterol, and blood pressure, can account for no more than 25–30% of excess cardiovascular risk factors in diabetic patients. This suggests that other factors might play a key role in the progression of atherosclerosis in diabetes. One of them is the disturbance of lipid metabolism. Atherothrombotic changes and high serum NEFA frequently accompany type 2 diabetic patients (5).

Some previous investigators emphasized the importance of the relationship between fatty acids and atherosclerosis. Hoak et al. (17) found thrombosis to be associated with the mobilization of fatty acids. Botti et al. (18) found that long-chain saturated fatty acids promote clotting. Connor et al. (19) reported the induction of fatal occlusive thrombi within minutes of infusing fatty acids. Michaud et al. (4) recently demonstrated that fatty acids enhance LPL production in human macrophage. It has been demonstrated that LPL secreted from macrophage contributes to the development and progression of atherosclerosis (3). Thus, fatty acids seem to participate in the development and progression of atherosclerosis in type 2 diabetic patients. To the best of our knowledge, however, the relationship between serum NEFA and atherosclerosis has not been examined in diabetic patients. In this respect, a major problem is that plasma glucose per se enhances LPL production in human macrophage and is also associated with atherosclerosis (6). In addition, it is well known that the degree of overweight and/or hypertension per se affects atherosclerosis. Therefore, we investigated NEFA level in nonobese nonhypertensive well-controlled unique type 2 diabetic patients (mean HbA1c 7.0%) and studied the relationship between atherosclerosis and serum NEFA level. As an index of atherosclerosis, we evaluated IMT in the plaque-free segments and carotid stenosis (% stenosis) in the segments of plaque using high-resolution B-mode ultrasound scan. This is the first description of the effect of serum NEFA on carotid atherosclerosis in type 2 diabetic patients.

In this study, we first demonstrated that serum NEFA is associated with both IMT in plaque-free segments and the degree of carotid stenosis in plaque segments in type 2 diabetic patients. However, the effect of NEFA on IMT in plaque-free segments and the degree of carotid stenosis were different. Whereas age was independently associated with IMT in plaque-free segments, NEFA was independently associated with the degree of atherosclerotic plaque. Thus, levels of circulatory NEFA may predict the degree of carotid atherosclerotic plaque in nonobese nonhypertensive well-controlled unique Japanese type 2 diabetic patients.

Presently, the mechanism by which serum NEFA level affects the degree of carotid atherosclerotic plaque in our unique Japanese type 2 diabetic patients is unknown. Although macrophages are able to use glucose, glutamine, and fatty acids as energy sources (20), complete oxidation of glucose and glutamine is limited in macrophages. Thus, fatty acids may constitute the crucial fuel for activated macrophage energy expenditure in the energy-limited environment of the atherosclerotic plaque.

Coagulation abnormalities are proposed as a further potentially important pathophysiological link between type 2 diabetes and atherosclerosis. In this respect, the study by Didisheim et al. (21), who showed that saturated long-chain fatty acids activate Hageman factor (factor XII), is very interesting. Hageman factor initiates the cascade sequence of enzymatic reactions, culminating in the production of thrombin and the conversion of fibrinogen to fibrin. Thrombin also induces an increase in fibrinogen biosynthesis. Pilgeram and Pickart (22) have shown that free fatty acids (FFAs), which are unbound by protein, stimulate the rate of biosynthesis of fibrinogen in vitro. Thereafter, Schneider et al. (23) demonstrated that FFAs had synergistic effects on insulin-stimulated increase in plasminogen activator inhibitor 1 (PAI-1) in the blood of type 2 diabetic patients. Decreased fibrinolytic capacity caused by overexpression of PAI-1 may also be related to NEFA-induced atherosclerosis in our diabetic patients. Kwok et al. (24) recently reported that linoleic acid and oleic acid increased the endothelin-1 binding and action in cultured rat aortic smooth muscle cells.

Finally, we could not find an association between carotid atherosclerosis and conventional risk factors, including LDL cholesterol, in our unique populations. The reason is unclear, but it may mean that the diabetic state per se is such a powerful factor on the carotid atherosclerosis that the effect of other risk factors is masked (25). Alternatively, it could be the result of the selection of diabetic subjects, because we excluded patients with known obesity, hypertension, cardiovascular disease, or ischemic stroke (26). Mohan et al. (25) recently demonstrated that diabetes and age, but not conventional risk factors, are the most important risk factors associated with increased IMT in South Indian diabetic patients with a BMI of 24.5 kg/m2.

In summary, although our cross-sectional study was performed with a limited number of patients (n = 54), it can be concluded that levels of serum NEFAs may predict the degree of carotid atherosclerotic plaque in nonobese nonhypertensive well-controlled unique type 2 diabetic patients. Prospective studies should be undertaken to confirm the validity of our findings.

The authors acknowledge Drs. Hajime Mizutani, Takahide Okumura, and Hiroyuki Kishimoto from the Division of Diabetes, Kansai Denryoku Hospital for their help in our research.

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Address correspondence and reprint requests to Ataru Taniguchi, MD, First Department of Internal Medicine, Kansai-Denryoku Hospital, 2-1-7 Fukushima, Fukushima-ku, Osaka-City, Osaka 553-0003 Japan. E-mail: K-58403@kepco.co.jp.