Serum Nonesterified Fatty Acids Are Increased in Nonobese Japanese Type 2 Diabetic Patients With Microalbuminuria

The major clinical consequence of type 2 diabetes is mortality and morbidity from atherosclerotic vascular disease. The degree of atherosclerosis can be evaluated by high-resolution B-mode ultrasound scan (1). Using high-resolution B-mode ultrasound scan, we recently demonstrated that serum nonesterified fatty acids (NEFAs) are independently associated with carotid atherosclerosis in nonobese nonhypertensive Japanese type 2 diabetic patients (2). Michaud et al. (3) recently reported that fatty acids enhance lipoprotein lipase (LPL) production in human macrophages. LPL secreted from macrophages is known to contribute to the development and progression of atherosclerosis (4). Thus, fatty acids are considered to be associated with atherosclerotic vascular disease in type 2 diabetic patients.

Slightly increased urinary albumin excretion rate, microalbuminuria, is considered as an index of diabetic nephropathy. Microalbuminuria has also been identified as an independent risk factor for atherosclerosis in type 2 diabetic patients. An association between microalbuminuria and cardiac disease in type 2 diabetic patients has been demonstrated (5). To the best of our knowledge, however, it remains to be clarified whether type 2 diabetic patients with microalbuminuria have higher concentrations of serum NEFAs than those with normoalbuminuria. In this context, a major problem is that microalbuminuria has been associated with atherosclerotic risk factors, such as high blood pressure, hypertriglyceridemia, and low HDL cholesterol, thus complicating the relation of microalbuminiuria with serum NEFAs (6,7). Moreover, it is well recognized that being overweight or hyperglycemic per se affects microalbuminuria and serum NEFA levels in humans. We therefore recruited nonobese type 2 diabetic patients with microalbuminuria who were carefully matched for BMI, blood pressure, serum lipid level, and fasting glucose to those with normoalbuminuria. This is the first description that serum NEFAs are increased in nonobese Japanese type 2 diabetic patients with microalbuminuria.

A total of 21 diabetic patients with microalbuminuria (15 men and 6 women) and 45 patients with normoalbuminuria (33 men and 12 women) were enrolled in the study. They all were nonobese (BMI <27 kg/m²) Japanese type 2 diabetic patients (8). Type 2 diabetes was diagnosed based on the criteria of the World Health Organization (9). All subjects had ingested at least 150 g carbohydrate for the 3 days preceding the study. A total of 14 (67%) patients with microalbuminuria and 23 (51%) patients with normoalbuminuria were taking sulfonylureas. The rest were treated with diet alone. None had received insulin therapy. Ten (48%) patients with microalbuminuria and 15 (33%) patients with normoalbuminuria were treated with antihypertensive drugs. Four (19%) patients with microalbuminuria and seven (16%) patients with normoalbuminuria were treated with lipid-lowering agents. There was no significant difference in sex and medication status between the patients with microalbuminuria and those with normoalbuminuria. They did not consume alcohol or perform heavy exercise for at least 1 week before the study.

Blood was drawn on the morning after a 12-h fast. Plasma glucose was measured with the glucose oxidase method. Total, HDL, and LDL cholesterol, triglycerides, remnant-like particle cholesterol (RLP-C), and NEFAs were also measured. The LDL cholesterol level was calculated using the Friedewald formula (10). RLP-C level was measured by the method reported by Nakajima et al. (11). Serum NEFAs were measured in duplicate using the enzymatic method (NEFA HR kit; Wako Chemicals, Osaka, Japan), and the mean of the two values was used (2). The coefficient of variation for NEFAs was 2%.

Urinary albumin concentration was assessed in a morning spot urine sample using the single radial immunodiffusion method (12). Urinary albumin concentration was measured in duplicate, and the mean of the two values was used for the study. Intra- and interassay coefficients of variations were <6% (Alb-Tia Seiken; Denka-Seiken, Tokyo, Japan). Several reports have indicated that early morning spot urine is usually sufficient for detecting the presence of microalbuminuria (13,14). In the present study, we calculated urinary albumin excretion rate as a ratio of urinary albumin and urinary creatinine, a ratio that markedly enhances the accuracy of the single spot urine sample in the assessment of microalbuminuria (15). Microalbumiuria was defined as a urinary albumin concentration >30 but <300 mg/g creatinine. Normoalbuminuria was defined as urinary albumin concentration <30 mg/g creatinine.

The statistical analysis was performed with the StatView 5.0 system (Statview, Berkeley, CA). The differences of mean were determined by Student’s t test. Data were expressed as the means ± SEM.

The clinical characteristics and profile between the patients with microalbuminuria (n = 21) and normoalbuminuria (n = 45) were compared. Urinary albumin concentrations in the patients with microalbuminuria and normoalbuminuria were 89 ± 12 mg/g creatinine (range 44–236) and 5 ± 1 (range 0.1–17.0), respectively. No significant difference was observed in age (64.2 ± 2.2 vs. 61.8 ± 1.0 years, P = 0.127), BMI (22.7 ± 0.4 vs. 22.8 ± 0.3 kg/m², P = 0.481), or systolic (128 ± 2 vs. 126 ± 2 mmHg, P = 0.288) and diastolic (73 ± 2 vs. 73 ± 1 mmHg, P = 0.448) blood pressure between the two groups. Fasting glucose (154 ± 7 vs. 143 ± 5 mg/dl, P = 0.109) and HbA_1c (7.5 ± 0.2 vs. 7.0 ± 0.2%, P = 0.066) levels were higher in patients with microalbuminuria than in those with normoalbuminuria, but the difference was not statistically significant. The patients with microalbuminuria had higher triglycerides (127 ± 13 vs. 110 ± 5 mg/dl, P = 0.066) and higher RLP-C (5.3 ± 0.5 vs. 4.9 ± 0.2 mg/dl, P = 0.208) concentrations than those with normoalbuminuria, but the difference was not statistically significant. There was no significant difference in serum total (200 ± 7 vs. 195 ± 5 mg/dl, P = 0.290), HDL (51 ± 3 vs. 50 ± 2 mg/dl, P = 0.279), and LDL cholesterol (124 ± 4 vs. 124 ± 6 mg/dl, P = 0.478) levels between the two groups. In contrast, serum NEFAs were significantly higher in patients with microalbuminuria (0.64 ± 0.05 mEq/l) than in those with normoalbuminuria (0.51 ± 0.02 mEq/l, P = 0.002).

In the present study, we first confirmed the presence of elevated NEFAs in nonobese Japanese type 2 diabetic patients with microalbuminuria. Microalbuminuria has been shown to be not only an indicator of incipient nephropathy, but also a risk marker for early mortality, especially in type 2 diabetes, due to cardiovascular diseases.

Although the mechanisms underlying the relation between NEFAs and microalbuminuria are unclear, serum NEFAs seem to be associated with early atherosclerotic changes in nonobese Japanese type 2 diabetic patients. Using high-resolution B-mode ultrasound scan, we very recently demonstrated that serum NEFAs are independently associated with carotid atherosclerosis in nonobese, nonhypertensive, well-controlled (mean HbA_1c 7.0%), unique type 2 diabetic patients (2). Interestingly, the degree of carotid stenosis was 8.1 ± 2.1%, suggesting that serum NEFAs are associated with the very early stages of carotid atherosclerosis. Microalbuminuria is also considered to be a risk marker for the early events in athrosclerosis. Thus, it may be hypothesized that serum NEFA level is reflective of the early stage of atherosclerosis in nonobese Japanese type 2 diabetic patients.

The mechanism by which serum NEFAs are associated with atherosclerosis in nonobese Japanese type 2 diabetic patients is unclear. In this respect, the recent report of Michaud et al. (3) that fatty acids enhance macrophage LPL production is very interesting. LPL secreted from macrophage is known to contribute to the development and progression of atherosclerosis (4). Alternatively, serum NEFAs might cause atherosclerosis by stimulating a coagulation cascade sequence. Didisheim et al. (16) previously showed that saturated long-chain fatty acids activate Hageman factor (factor XII). 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 (17) have shown that free fatty acids (FFAs) stimulate the rate of biosynthesis of fibrinogen in vitro. Schneider et al. (18) demonstrated that FFAs had synergistic effects on the insulin-stimulated increase in plasminogen activator inhibitor-1 (PAI-1) in the blood of type 2 diabetic patients. Enhanced coagulation cascade sequence in conjunction with decreased fibrinolytic capacity due to overexpression of PAI-1 might explain the reason why serum NEFA level is associated with the early stage of atherosclerosis and microalbuminuria in nonobese Japanese type 2 diabetic patients.

In summary, we first demonstrated that serum NEFAs are increased in nonobese Japanese type 2 diabetic patients with microalbuminuria. Further study should be undertaken to clarify whether serum NEFAs are reflective of the early stages of atherosclerosis, including microalbuminuria, in nonobese Japanese type 2 diabetic patients.