Effects of C-peptide on Adipocytes and Mesenchymal Stem Cells in Human Adipose Tissue Ahmad Ghorbani1, Mohammad Taghi Rajabi-Mashhadi2, Seyyed Abbas Zojaji1, Reza Shafiee-Nick1 1Department of Pharmacology & Pharmacological Research Center of Medicinal Plants, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2Endoscopic & Minimally Invasive Surgery Research Center, Mashhad University of Medical Sciences, Mashhad, Iran ABSTRACT Recent studies suggest that proinsulin connecting peptide (C-peptide) may show physiological functions in various tissues. This study was aimed to determine whether C-peptide could be involved in the regulation of lipolysis, adiponectin release, and function of mesenchymal stem cells (MSCs) in adipose tissue. Human subcutaneous adipose tissue was cultured in the presence of C-peptide and lipolysis was determined by glycerol measurement in the conditioned media. Effect of C-peptide on adiponectin secretion was evaluated in differentiated adipocytes. The adipogenic and osteogenic abilities of adipose MSCs were evaluated using Oil Red and Alizarin Red staining, respectively. C-peptide induced a significant decrease in basal lipolysis, a slight decrease (statistically insignificant) in isoproterenol-stimulated lipolysis, and a slight increase in adiponectin secretion at concentration of 8 nM, 16 nM and 4 nM, respectively. It had no effect on adipogenic and osteogenic differentiation of MSCs. However, at concentration of 4 nM, C-peptide significantly increased the proliferative capability of MSCs (p<0.05). These results suggest that C-peptide has physiological effects in human subcutaneous adipose tissue and contributes to the regulation of basal lipolysis and pool of MSCs. INTRODUCTION Recent data suggest that proinsulin C-peptide may exhibit characteristics of a peptide hormone and show physiological functions in various tissues. Diabetic patients show a spectrum of abnormalities in lipid metabolism including uncontrolled lipolysis, and dysregulation of adipogenesis and lipogenesis in adipose tissue. These abnormalities are associated with development of atherosclerosis and cardiovascular diseases. Also, there are some reports that diabetes may impair mobilization ability and wound healing capability of mesenchymal stem cells (MSCs) in the patients with type 1 and type 2 diabetes. Until now, the effects of C-peptide on adipose tissue are not yet well known. The aim of this study was to examine whether CP could be involved in the regulation of lipolysis, adiponectin secretion, and proliferation and differentiation abilities of MSCs in human adipose tissue. MATERIALS AND METHODS Subcutaneous adipose tissue samples were obtained from patients undergoing conventional intra- abdominal surgery. Effect of C-peptide on lipolysis was evaluated in ex-vivo organ culture condition and the level of lipolysis was determined by measurement of glycerol. In separate experiments, MSCs were isolated from adipose tissue samples using collagenase-based technique. Effect of C-peptide on proliferation of MSCs was evaluated using MTT assay. The adipogenic and osteogenic differentiation abilities of adipose MSCs were evaluated using Oil Red and Alizarin Red staining, respectively. Effect of C-peptide on adiponectin secretion was evaluated in differentiated adipocytes. RESULTS C-peptide significantly reduced the level of basal lipolysis (p < 0.05) and showed a slight inhibitory effect (statistically insignificant) on isoproterenol-stimulated lipolysis (Fig. 1). Flow cytometric analysis showed that the MSCs were positive for stem cell-associated markers (CD44 and CD105) and negative for haematopoietic markers (CD34 and CD45). C-peptide at concentrations of 1-16 nM increased proliferation of MSCs (Fig. 2). The effect of C-peptide at 4 nM was statistically significant. Figure 3 shows that C-peptide at concentrations of 4-16 nM had no effect on the level of osteogenic differentiation. Figure 4 demonstrates that