A total of 126 type 2 diabetic participants and 37 healthy controls were enrolled in the study. All participants had been treated at an outpatient clinic and were recruited over 12 months. The healthy controls were chosen from an epidemiologic study conducted at the Ansan Cohort Center, which was held in 1998 in Ansan-si, Gyeonggi-do, South Korea. For the control group, age, and sex-matched participants who had no known disease history and no abnormal laboratory findings were chosen. The diagnosis of type 2 diabetes mellitus was made according to the Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus [14]. Participants had to fulfill three additional criteria for inclusion: no episode of ketoacidosis, no ketonuria, and insulin therapy initiated after at least 5 years of known disease. Participants with non-diabetic kidney diseases, and participants with azotemia (serum creatinine >1.6 mg/dL) were excluded. Participants were instructed not to make lifestyle changes or changes to their oral hypoglycemic or/and insulin therapy during the study period. Ethical approval was obtained from the Korea University Institutional Review Board, and written informed consent was obtained from all study participants.

Among the diabetic participants, 74 had hypertension. All hypertensive diabetic participants were being treated with antihypertensive medication, 61 participants were taking an angiotensin-converting enzyme inhibitor or an angiotensin II receptor antagonist with or without other antihypertensive medications. The other 13 participants who were not receiving a reninangiotensin system (RAS) blockade were being treated with calcium channel blockers, or alpha or beta blockers. Venous blood was taken from all participants after overnight fasting. Fasting blood glucose and postprandial 2-hour glucose levels were measured using the hexokinase method. Total protein, albumin, hemoglobin, creatinine, total cholesterol, triglyceride, hsCRP, and high-density lipoprotein (HDL) cholesterol levels were also measured. Hemoglobin A1c (HbA1c) was measured by high performance liquid chromatography (HPLC). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the standard formula. A 24-hour urine collection was performed for the determination of urinary albumin excretion and creatinine clearance. Urinary albumin excretion was measured by radioimmunoassay (Immunotech, France) at a sensitivity of 0.5 mg/L. Plasma and urinary creatinine levels were measured using the modified Jaffe method. Creatinine clearance was calculated using the morning creatinine level and was standardized for a body surface area using the standard formula. Plasma MCP-1 and leptin levels and secreted MCP-1 from culture supernatants were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). The diabetic participants were divided into three groups: a normal albuminuric diabetic group with a 24-hour urinary albumin excretion (UAE) lower than 30 mg in two or more urine samples and no more than one value greater than or equal to 30 mg (n=40); a microalbuminuric diabetic group with a 24-hour UAE in the range of 30–299 mg in at least two urine samples (n=41); and an overt proteinuria group of proteinuric diabetic participants, defined as having a 24-hour UAE greater than or equal to 300 mg (n=35).

A rat vascular smooth muscle cell line (CRL-2018) was obtained from ATCC (Manassas, VA, USA). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 u/mL antibiotics at 37 °C under a humidified 5% CO₂ atmosphere. Cultures were fed every 2–3 days during growth and every 2 days after confluence. Cells were used at 10 passages in this study. VSMCs were cultivated on 100 mm dishes and serum-restricted for 24 hours to determine whether leptin could directly increase MCP-1 synthesis. Afterward, different concentrations of recombinant rat leptin (Sigma-Aldrich, St. Louis, MO, USA) were added to culture media at final concentrations of 1 ng/mL, 10 ng/mL, and 100 ng/mL. Three or 12 hours later, the media were collected, and the cells were scraped from the dishes in the presence of extraction buffer (20 mM Tris–HCl, pH 7.4, 10 mM ethylenediaminetetraacetic acid, 5 mM ethylene glycol tetraacetate, 5 mM β-mercaptoethanol, 50 μg/mL phenylmethyl sulfonyl fluoride, 10 mM benzamidine, and 0.1 μg/mL aprotinin) and homogenized. Cell lysates and conditioned media were centrifuged at 15,000 rpm for 10 min at 4 °C, and the supernatants were collected. To define the mechanism of leptin-mediated MCP-1 synthesis and whether or not it is mediated by the MAPK/extracellular signal–regulated kinase (ERK) pathway, a selective MAPK kinase (MEK) inhibitor, PD98059 (Sigma-Aldrich, St. Louis, MO, USA), was added to the cells at a concentration of 10 μM 1 hour before treatment with leptin. Next, we performed another experiment to determine the effects of high glucose and leptin stimulation on the MAPK signaling pathway. Subconfluent VSMCs were cultured for 24 hours in a medium containing 5 mmol/L or 30 mmol/L of D-glucose. The cells were then treated with leptin at final concentrations of 1 ng/mL, 10 ng/mL, and 100 ng/mL, and harvested after 6 hours. In addition, we investigated whether leptin stimulation induces the sequential activation of the MAPK pathway, as occurs with the MEK, MAPK (ERK), and E26 (ETS)-like transcription factor (ELK) pathways. Since leptin increased maximal MCP-1 production at a final concentration of 100 ng/mL, leptin was added to the VSMCs at final concentrations of 100 ng/mL, and cells were harvested at 30 minutes, 1 hour, 3 hours, 6 hours, and 12 hours, and then the proteins were extracted. To avoid any confounding effects of serum on the MAPK pathway, all experiments were performed in serum-free media. All experimental groups were cultured in triplicate. The results were representative of those from three independent experiments.

VSMCs were resuspended in Trizol reagent (Invitrogen, Carlsbad, CA, USA) containing RNase inhibitor. Reverse transcription (RT) was performed using an RT polymerase chain reaction (PCR) kit (PerkinElmer, Foster City, CA, USA). A total of 1 μg of RNA and oligo-(dT)12 primers were used. The reaction mixture was incubated for 60 min at 42 °C, and was then heated for 7 min at 90 °C in a thermocycler (Crocodile III, Oncor Co., Japan). After cDNA synthesis by RT, PCR amplification was done. For MCP-1, the sense primer was 5′ GAC CTG TTT GTC CGT AAG GC 3′, and the antisense primer was 5′ GAC CTG TTT GCA ACG GGC TG 3′. The mixture was heated at 94 °C for 30 seconds, at 55 °C for 30 s, at 72 °C for 30 seconds, and this was repeated by 38 cycles. For ERK1/2, the sense primer was 3′ CTT CCT CTA CCA GAT CCT CC 5′, and the antisense primer was 3′ GTC AAG AGC TTT GGA GTC AG 5′. The mixture was heated at 94 °C for 1 min, at 57 °C for 1 min, at 72 °C for 1 min, and this was repeated by 36 cycles. The PCR data were expressed in relative values as MCP-1/β-actin, Erk/β-actin.

Cells were lysed in lysis buffer. The protein was electrophoresed on a 10% SDS–PAGE minigel under denaturing conditions. The protein was transferred onto a nitrocellulose membrane. Then, separate reactions were conducted with rabbit anti-MEK antibody, anti-p-MEK antibody, anti-ERK1/2 antibody, anti-p-ERK1/2 antibody, anti-ELK antibody, and anti-p-ELK antibody (New England Biolabs, Inc., Beverly, MA, USA) diluted 1:1000 applied to the membrane. The filter was then washed four times with PBST and incubated with horseradish peroxidase-conjugated secondary antibody diluted 1:1000 for 60 min at room temperature. The detection of specific signals was performed using the ECL method. Equal amounts of protein loading were confirmed by Coomassie blue staining of the gel.

For parametrically distributed data, we used Student's unpaired t-tests and analysis of variance (ANOVA) for comparisons of quantitative variables, and chi-square tests for comparisons of proportions. Plasma leptin, MCP-1, and UAE levels were positively skewed with a high frequency of low values. Because data transformation would not have normalized this distribution, we used non-parametric statistical methods. For the analysis of in vitro data, a nonparametric analysis was used due to the small sample. A Kruskall-Wallis test was used for comparison of more than two groups, followed by a Mann-Whitney U test, using a microcomputer-assisted program with SPSS for Windows 10.0 (SPSS Inc., Chicago, IL, USA). Correlations between plasma leptin levels and biochemical parameters were examined using Spearman's rank correlation and by stepwise multiple regression analysis. P values less than 0.05 were considered statistically significant. Results were expressed as mean±SEM.

Table 1 shows the baseline clinical characteristics of the study sample. There were no significant differences between the participants and controls for age, sex, body weight, hemoglobin, albumin, and creatinine levels. These parameters were similar among the diabetic participants based on nephropathy status. There was no significant difference in the proportion of patients with previous history of cardiovascular disease or cerebrovascular diseases among diabetic patients (Table 1). The control group had a lower mean body mass index (BMI), fasting plasma glucose, postprandial 2-hour plasma glucose, total cholesterol, triglyceride, and hsCRP levels, systolic and diastolic blood pressure, UAE rate, and higher creatinine clearance than the diabetic groups. Among the diabetic participants, there were no significant differences in body weight, BMI, or systolic and diastolic blood pressure. The overt proteinuria group had higher UAE values and lower creatinine clearance than the normal and microalbuminuria groups. The normal albuminuric group showed higher levels of fasting plasma glucose and postprandial 2-hour plasma glucose levels than the microalbuminuria and overt proteinuria groups.

Plasma leptin levels were markedly elevated in the type 2 diabetic participants compared to the controls even in the normal albuminuric group (Table 1). Interestingly, plasma leptin levels were significantly higher in the overt proteinuria group than in the normal and microalbuminuria groups. Plasma MCP-1 concentrations were also significantly higher in the diabetic group than the control group. In the diabetic group, plasma MCP-1 levels increased according to the state of diabetic nephropathy. Since median value of plasma leptin level was 30 ng/mL, we reanalyzed the plasma MCP-1 levels based on the plasma leptin concentration using a cutoff value of 30 ng/mL. When we compared MCP-1 levels after adjusting for the four groups according to plasma leptin levels, participants who had leptin levels over 30 ng/mL had showed markedly higher levels of MCP-1 than participants with leptin levels below 30 ng/mL (Fig. 1). Table 2 shows the correlation analysis results between plasma leptin levels and various clinical parameters in the study participants. Plasma leptin levels were positively correlated with body mass index, fasting and postprandial blood glucose, HbA1c, total cholesterol, UAE, hsCRP, and plasma MCP-1 levels, and negatively correlated with creatinine clearance. However, plasma leptin concentrations did not show a significant relationship with other risk factors, including body weight, triglyceride levels, creatinine values, and systolic and diastolic blood pressure. By stepwise multiple regression analysis, plasma MCP-1 levels as a dependent variable at a cutoff value of 100 pg/mL, plasma leptin levels was only included in the model (betas±SE=−0.048±0.001, P<0.001).

Since plasma leptin levels appear to be the most significant independent factor for MCP-1 plasma levels, we next observed the effect of leptin stimulation on MCP-1 production. As shown in Fig. 2, MCP-1 mRNA expression significantly increased after leptin stimulation in a concentration-dependent manner, except at 1 ng/mL concentration. Interestingly, selective MEK inhibitor PD98059, significantly inhibited leptin-induced MCP-1 gene expression. Based on gene expression pattern, secreted MCP-1 protein also showed a similar tendency (Fig. 2). MCP-1 secretion also time-dependently increased from 3–12 hours, and prior treatment with PD98059 also showed a significant decrease in MCP-1 production (Fig. 2).

Since MEK inhibitor PD98059 significantly suppressed leptin-induced MCP-1 production, we further evaluated whether MCP-1 production induced by leptin depends on the MAPK pathway. We first examined the activation of MAPK (ERK) in response to leptin stimulation. As shown in Fig. 3, leptin stimulation significantly increased ERK gene expression. Additionally, the activation of ERK, assessed by measuring the levels of phospho-specific ERK, was found to increase in response to leptin. However, we did not detect dose-dependent activation of ERK by leptin stimulation (Fig. 3).

We next examined the effect of leptin on the activation of MEK, which is an upstream activator of MAPK. Phospho-specific MEK, an indication of MEK activation, occurred in response to leptin stimulation. Similar to ERK activation, we could not find a dose-dependent activation of MEK by leptin stimulation; however, there was no significant difference in total MEK protein expression among the groups (Fig. 4). In addition, we determined whether activation of the MEK-MAPK pathway could induce a parallel increase in the activity of a MAPK target transcription factor, ELK. As shown in Fig. 4, ELK activation measured by the level of phospho-specific ELK was also observed after leptin stimulation. However, no change in total ELK protein expression was observed based on the concentration of leptin.

Finally, we determined whether leptin stimulation induced a sequential activation of the MAPK pathway, we examined the activation of MEK, ERK, and ELK in response to leptin at a final concentration of 100 ng/mL, and harvested after 30 minutes, 1 hour, 3 hours, 6 hours, and 12 hours. As shown in Fig. 5, the activation of MEK was found to rapidly increase in response to leptin after 30 min, whereas maximal activation of ERK and ELK was observed at the 3- and 6-hour intervals, respectively.