Abstract
ObjectiveWe aimed to explore the role and possible mechanism of leptin in lower-extremity artery calcification in patients with type 2 diabetes mellitus (T2DM).MethodsWe recruited 59 male patients with T2DM and 39 non-diabetic male participants. All participants underwent computed tomography scan of lower-extremity arteries. The calcification scores (CSs) were analyzed by standardized software. Plasma leptin level was determined by radioimmunoassay kits. Human vascular smooth muscle cells (VSMCs) calcification model was established by beta-glycerophosphate and calcium chlorideinduction. Calcium deposition and mineralization were measured by the o-cresolphthalein complexone method and Alizarin Red staining. The mRNA expression of bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) was determined by quantitative RT-PCR. The protein levels of BMP2, Runx2, α-smooth muscle actin (α-SMA) and (p)-Akt was determined by Western-blot analysis, and α-SMA was also measured by immunofluorescence analysis.ResultsCompared with controls, patients with T2DM showed higher median calcification score in lower-extremity artery [286.50 (IQR 83.41, 1082.00) vs 68.66 (3.41, 141.30), p<0.01]. Plasma leptin level was higher in patients with calcification score ≥300 than ≥100 (252.67 ± 98.57 vs 189.38 ± 44.19 pg/ml, p<0.05). Compared with calcification medium, intracellular calcium content was significantly increased in VSMCs treated by leptin (200, 400 and 800 ng/ml) combined with calcification medium [11.99 ± 3.63, 15.18 ± 4.55, and 24.14 ± 5.85 mg/ml, respectively, vs 7.27 ± 1.54 mg/ml, all p<0.01]. Compared with calcification medium, Alizarin Red staining showed calcium disposition was more obvious, and the mRNA level of BMP2, Runx2 and OCN was significantly increased, and immunofluorescence and Western blot analysis showed that the expression of α-SMA was downregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium, respectively. Compared with calcification medium, the protein level of BMP2 and Runx2 was upregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium. Moreover, blocking PI3K/Akt signaling pathway can decrease the protein expression of BMP2 and Runx2 in VSMCs treated by leptin (400 ng/ml) combined with calcification medium.ConclusionsLeptin promoted lower-extremity artery calcification of T2DM by upregulating the expression of BMP2 and Runx2, and regulating phenotypic switch of VSMCs via PI3K/Akt signaling pathway.
Highlights
The global prevalence of diabetes in adults has increased in recent decades
Hyperglycemia and increased glycation end products, hyperinsulinemia, oxidative stress, lipid metabolism disturbance, inflammation, apoptosis and abnormal expression of bone regulatory protein are the main factors of vascular calcification in type 2 diabetes mellitus (T2DM), which are closely related to the occurrence and development of vascular calcification [31, 32]
A high baseline level of leptin was found associated with increasing coronary artery calcium severity [36]
Summary
The global prevalence of diabetes in adults has increased in recent decades. The number of patients 20 to 79 years old with diabetes is predicted to increase to 642 million by 2040 [1]. Adipose tissue is metabolically active and produces a multitude of adipokines, which may direct effects on the skeleton as well as on vascular structure and function [4]. Clinical studies suggested that adipose tissue plays an important role in the formation of vascular calcification (VC) [7,8,9]. The central function of leptin is to inhibit food intake and appetite, and the peripheral effects are mainly related to insulin resistance, inflammatory response, oxidative stress, atherosclerosis [11, 12]. Endothelial dysfunction, abnormal mineral metabolism and increased inflammatory factors are involved in the process of vascular calcification [16]. Previous studies have found that adipokines play an important role in vascular calcification, and confirmed that leptin regulates the osteoblastic differentiation and calcification of vascular cells [17]. Leptin is associated with insulin resistance and demonstrated as a significant independent predictor of coronary artery calcification [18]
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