Leptin, triglycerides, and interleukin 6 are independently associated with C-reactive protein in Japanese type 2 diabetic patients

1. Introduction 

The major clinical consequence of type 2 diabetes is mortality and morbidity from atherosclerotic vascular disease especially coronary heart disease (CHD). As regards to the risk factors responsible for the evolution of atherosclerosis in diabetic patients, Bierman [1] previously estimated that typical risk factors including smoking, cholesterol, and blood pressure can account for no more than 25–30% of excess cardiovascular risk factor in diabetic patients. This suggests that other factors might play a key role in the progression of atherosclerosis in diabetes.

Whereas insulin resistance is established to be one of the risk factors for the evolution of CHD [2], there are some data suggesting that subclinical inflammation is associated with CHD events in man [3], [4], [5], [6], [7]. Elevated levels of C-reactive protein (CRP), although still for the most part in the healthy reference range, have been shown to be associated with increased risk of future CHD events [3], [4], [5]. Some cross-sectional and case-control studies have reported elevated antibody titers directed against Chlamydia pneumoniae, Helicobacter pyroli, and Cytomegalovirus among those with prevalent heart disease [6]. We recently presented a prospective analysis of the factors in relation to the incidence of CHD during 4.5 years of follow-up and found that insulin resistance and low-grade inflammation are independently associated with the development of CHD events in Japanese type 2 diabetic patients [7].

Leptin is the neuroregulatory peptide secreted from adipose tissue [8]. In addition to be an “adiposity signal” for the long-term regulation of body weight, leptin seems to have a potential signal leading to the increased risk of vascular events in humans. It is shown that leptin correlates with other risk factors responsible for the evolution of atherosclerosis such as apoprotein B and systolic blood pressure, independent of BMI and glucose disposal rate [9]. Wallace et al. [10] have recently demonstrated in a large prospective study that leptin is a novel independent risk factor for CHD in humans. High leptin levels are shown to predict subsequent development of type 2 diabetes in Japanese-Americans [11].

To the best of our knowledge, however, the relationship between CRP and leptin was not yet investigated in type 2 diabetic patients. Macrovasular disease per se is considered to affect the levels of CRP and leptin in diabetic patients. We therefore recruited Japanese type 2 diabetic patients who had no evidence of cardiovascular disease, ischemic stroke, or chronic renal failure and investigated the relationships between CRP and leptin.

2. Subjects and methods 

About 148 Japanese type 2 diabetic patients who visited Kansai-Denryoku Hospital were enrolled for the present study. Type 2 diabetes mellitus was diagnosed based on the criteria of WHO [12]. They had no evidence of current acute illness including clinically significant infectious disease. The duration of diabetes was 10.8±0.6 yr (mean±S.E.M.) (range; 1–35 yr). One-hundred and nineteen of 148 diabetic patients were taking sulfonylureas and the rest were treated with diet alone. No patients have received insulin therapy. All subjects had ingested at least 150g of carbohydrate for the 3 days preceding the study. None of the subjects had significant renal, hepatic, or cardiovascular disease. Patients did not consume alcohol or perform heavy exercise for at least one week before the study.

Blood was drawn at the morning after a 12h fast. Plasma glucose was measured with glucose oxidase method. The triglycerides, total cholesterol, and HDL cholesterol were also measured. Serum insulin was measured using a two-site immunoradiometric assay (Insulin Riabead II, Dainabot, Japan). CRP was measured by ultrasensitive competitive immunoassay (antibodied and antigens from Calbiochem) as described previously [13]. Serum IL-6 was measured by enzyme-linked immunosorbent assay (Quantikine IL-6, R&D Systems, Oxford, UK) [14]. Serum leptin concentration was measured with a radioimmunoassay kit (Linco Research, St Charles, MO) as described previously [15]. Samples for insulin, CRP, IL-6, and leptin were prepared, frozen, and stored at −70°C until the assay.

The estimate of insulin resistance by HOMA (HOMA-IR) was calculated with the formula: fasting serum insulin (μU/ml)×fasting plasma glucose (mmol/l)/22.5 [16]. It may be argued that the use of sulfonylureas in patients with diabetes might significantly affect the estimate of insulin resistance by HOMA, as these drugs are known to decrease fasting plasma glucose without substantially changing fasting plasma insulin [17]. It seems, however, unlikely because Bonora et al. [18] and Emoto et al. [19] showed that in the validation studies of HOMA, the correlation of insulin sensitivity estimated by such method and that measured by the glucose clamp was not substantially different in diet-treated and sulfonylurea-treated type 2 diabetes.

3. Data analysis 

Data were presented as means±S.E.M. Statistical analyses were conducted using the StatView 5 system (Statview, Berkeley, CA). Simple (Spearman's rank) correlation coefficient and stepwise multiple regression analyses were used to examine the relationships between CRP and IL-6, leptin, and the measures of variables including triglycerides and BMI. P<0.05 was considered as significant. In multivariate analyses, F-value ≥4 was considered as significant.

4. Results 

The subjects studied were all Japanese type 2 diabetic patients (109 men and 39 women) with an age range of 37–84 years (62.2±0.8 yr) and a BMI of 15.4–35.8kg/m2 (23.6±0.3kg/m2). The fasting plasma glucose was 143±3mg/dl (range, 76–263mg/dl) and glycosylated hemoglobin (HbA1c) was 7.1±0.1% (range 4.9–10.4%). Fasting insulin level was 7.0±0.3μU/ml (range 1.6–21.2μU/ml). Serum triglycerides, total and HDL cholesterol levels were 128±6 and 201±3, and 58±1mg/dl, respectively. Serum CRP, IL-6, and leptin concentrations were 788±49ng/ml (range, 50–2470ng/ml), 1.98±0.15pg/ml (range, 0.5–19.3pg/ml), and 5.6±0.4ng/ml (range, 1.1–23.4ng/ml), respectively.

Table 1 illustrates the correlation between CRP and IL-6, leptin, and the measures of variables including BMI and triglycerides in our diabetic patients. CRP value was positively correlated to IL-6 (r=0.323, P<0.001), leptin (r=0.330, P<0.001), BMI (r=0.281, P<0.001), triglycerides (r=0.293, P<0.001), HOMA-IR (r=0.294, P<0.001), and diastolic blood pressure (r=0.176, P=0.048). In contrast, CRP value was negatively correlated to HDL cholesterol level (r=−0.181, P=0.028). There was, however, no relationship between CRP and measures of variables including age, gender, duration of diabetes, or medication status.

Table 1. Correlation of CRP to measures of variables in diabetic patients

		r	P	F
	IL-6	0.323	<0.001	4.04
	Leptin	0.330	<0.001	7.09
	BMI	0.281	<0.001	2.44
	Triglyceride	0.293	<0.001	15.13
	HOMA-IR	0.294	<0.001	0.01
	HDL cholesterol	−0.181	0.028	0.89
	Total cholesterol	0.074	0.369	–
	Fasting glucose	−0.003	0.974	–
	HbA1c	−0.003	0.966	–
	Systolic blood pressure	0.084	0.342	–
	Diastolic blood pressure	0.176	0.048	0.31
	Age	−0.066	0.426	–
	Gender	−0.041	0.620	–
	Duration of diabetes	−0.117	0.163	–
	Therapy for diabetes	−0.122	0.143	–

Multiple regression analyses were performed using the stepwise procedure. The analyses included CRP as the dependent variable and candidate risk factors (IL-6, leptin, BMI, triglyceride, HDL-cholesterol, diastolic blood pressure) as independent variables. CRP was independently predicted by IL-6 (P<0.001, F=4.04), leptin (P<0.001, F=7.09), and triglycerides (P<0.001, F=15.13), which explained 17.6% of the variability of CRP in our diabetic patients. Other variables including BMI were not associated with CRP in our Japanese type 2 diabetic patients (Table 1).

5. Discussion 

Atherosclerotic vascular disease especially coronary heart disease (CHD) is a major cause of death among diabetic patients in many countries including Japan. The risk of CHD, however, appears to be similar in patients with type 2 diabetes and impaired glucose tolerance [20], [21]. Thus, the factors other than the level of glycemia seem to accelerate the development of CHD in type 2 diabetes. This idea is supported from the notion that the duration of diabetes and the level of glycemia are not the risk factors for atherosclerosis including CHD in type 2 diabetic patients [22], [23].

A number of literatures have shown that age, smoking, blood pressure, abnormalities of lipids or lipoprotein particles, and insulin resistance could lead to the development of atherosclerosis including CHD in humans. The assay of high sensitive CRP enabled us to make sure that low-grade inflammation might play a major role in the development of CHD in humans [2], [3], [4], [5], [6]. During 4.5 years of follow-up study, we demonstrated that insulin resistance and low-grade inflammation are independently associated with the development of CHD events in Japanese type 2 diabetic patients [7].

An association of human CHD with Chlamydia pneumonia, Cytomegalovirus, and periodontal disease favors the importance of low-grade inflammation in the development of CHD [6]. Saremi et al. [24] recently reported that among subclinical infections, periodontal disease is one of the most important risk factors for the death caused by cardiorenal disease in type 2 diabetic patients. Using IgG titer against Porphyromonas gingivalis, we previously showed that periodontal disease is associated with carotid atherosclerosis and albuminuria in Japanese type 2 diabetic patients [25], [26]. Carotid atherosclerosis and albuminuria are both considered being one of the important markers for the future development of CHD in type 2 diabetic patients. Thus, low-grade inflammation per se seems to play an important role in the evolution of atherosclerosis in type 2 diabetic patients.

However, the factors responsible for elevation of CRP were not yet fully clarified in type 2 diabetic patients. For that reason, we investigated the factors associated with CRP and found for the first time that in conjunction with IL-6 and triglycerides, leptin is another important factor that is associated with the elevation of CRP in our diabetic patients.

Our results may be explained by the inflammation in adipose tissue. We previously demonstrated that in Japanese type 2 diabetic patients, the levels of serum leptin and triglycerides were positively correlated with subcutaneous and visceral fat areas, respectively [15], [27]. Recent report has shown that inflammatory cells such as monocytes and macrophages within the adipose tissue may contribute to the synthesis of IL-6 [28]. Yudkin et al. [29] showed that proinflammatory cytokines such as IL-6 and tumor necrosis factor-α play an important role in the low level of chronic inflammatory state.

In addition to being a signal for regulating body weight, leptin might signal an increased risk of vascular events in humans. Data from several different populations including ours have suggested a strong positive correlation between leptin and insulin concentrations, and insulin-resistant men have higher leptin concentrations than those who are insulin-sensitive, independent of body fat mass [15], [30], [31], [32]. Bastard et al. [32] showed that not only leptin but also IL-6 are associated with body mass index and insulin resistance and that IL-6 and leptin are interrelated in Caucasian obese type 2 diabetic patients. Leptin seems to correlate with other risk factors responsible for the evolution of atherosclerosis such as apoprotein B and systolic blood pressure, independent of BMI and glucose disposal rate [9]. Wallace et al. [10] have recently demonstrated that leptin is a novel independent risk factor for CHD. In vitro study has shown that leptin has a stimulatory effect on platelet aggregation induced by adenosine phosphate [33]. Bodary et al. [34] demonstrated that leptin contributes to arterial thrombosis following vascular injury in vivo and these prothrombotic effect appear to be mediated through the platelet leptin receptor in mice. Finally, our report is compatible with the report shown by Kazumi et al. [35] that fat mass and leptin, but not adiponectin were associated with CRP in young healthy men. Cleland et al. [36] recently showed the link between leptin and CRP in healthy subjects.

In summary, it can be concluded that in conjunction with serum IL-6 and serum triglycerides, serum leptin is another important independent factor associated with CRP in Japanese type 2 diabetic patients. Thus, our present study might suggest the possibility of pathophysiological mechanism by which leptin may play a fundamental role in the development of diabetic complications such as cardiovascular disease.