C-peptide reduces high-glucose-induced apoptosis of endothelial cells and decreases NAD(P)H-oxidase reactive oxygen species generation in human aortic endothelial cells

Abstract

Aims/hypothesis Reactive oxygen species (ROS) generated during hyperglycaemia are implicated in the development of diabetic vascular complications. High glucose increases oxidative stress in endothelial cells and induces apoptosis. A major source of ROS in endothelial cells exposed to glucose is the NAD(P)H oxidase enzyme. Several studies demonstrated that C-peptide, the product of proinsulin cleavage within the pancreatic beta cells, displays anti-inflammatory effects in certain models of vascular dysfunction. However, the molecular mechanism underlying this effect is unclear. We hypothesised that C-peptide reduces glucose-induced ROS generation by decreasing NAD(P)H oxidase activation and prevents apoptosis Methods Human aortic endothelial cells (HAEC) were exposed to 25 mmol/l glucose in the presence or absence of C-peptide and tested for protein quantity and activity of caspase-3 and other apoptosis markers by ELISA, TUNEL and immunoblotting. Intracellular ROS were measured by flow cytometry using the ROS sensitive dye chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H 2 -DCDFA). NAD(P)H oxidase activation was assayed by lucigenin. Membrane and cytoplasmic levels of the NAD (P)H subunit ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC-1) and its GTPase activity were studied by immunoblotting and ELISA. RAC-1 (also known as RAC1) gene expression was investigated by quantitative real-time PCR. Results C-peptide significantly decreased caspase-3 levels and activity and upregulated production of the antiapoptotic factor B cell CLL/lymphoma 2 (BCL-2). Glucose-induced ROS production was quenched by C-peptide and this was associated with a decreased NAD (P)H oxidase activity and reduced RAC-1 membrane production and GTPase activity. Conclusions/interpretation In glucose-exposed endothelial cells, C-peptide acts as an endogenous antioxidant molecule by reducing RAC-1 translocation to membrane and NAD (P)H oxidase activation. By preventing oxidative stress, C-peptide protects endothelial cells from glucose-induced apoptosis.

Physiological effects and therapeutic potential of proinsulin C-peptide

“…C-peptide reduces levels and activity of activated caspase-3 while increasing levels of the antiapoptotic molecule Bcl-2 in high-glucoseexposed endothelial cells (13). Similar antiapoptotic activities of C-peptide are reported in human neuroblastoma SH-SY5Y cells in the hippocampus of type 1 diabetes rats and in TNF-␣-exposed kidney proximal tubular cells (2,61,95).…”

“…A direct inhibitory effect of C-peptide on endothelial cell ROS formation is now documented from several laboratories (5,13,107). Stimulation and increased expression of both eNOS and Na ϩ ,K ϩ -ATPase activities in several tissues is also well documented (25,44,72,97,112), and an inhibitory effect on NF-B results in diminished diabetes-mediated expression of cytokines, chemokines, and cell adhesion molecules (12,57,71,89,104,106).…”

“…Stimulation and increased expression of both eNOS and Na ϩ ,K ϩ -ATPase activities in several tissues is also well documented (25,44,72,97,112), and an inhibitory effect on NF-B results in diminished diabetes-mediated expression of cytokines, chemokines, and cell adhesion molecules (12,57,71,89,104,106). Finally, C-peptide activates antiapoptotic mechanisms by inhibiting TG-2 and caspase-3 (13). Overall, C-peptide may protect against the deleterious effects of hyperglycemia by improving microcirculation, alleviating endothelial dysfunction, and inhibiting endothelial apoptosis; see Fig.…”

“…Increasing evidence suggests that a major biological activity of C-peptide in the periphery is its anti-inflammatory and antiapoptotic effects on vascular endothelial cells of different tissues as well as in neurons and kidney tubular cells affected by the deleterious effects of high glucose or TNF-␣ (2,5,13,61,90,95). Results from small clinical trials in which C-peptide was administered, together with insulin, to type 1 diabetic subjects suffering from nephropathy and neuropathy demonstrated C-peptide efficacy in amelioration of these complications, suggesting that maintenance of physiological levels of C-peptide prevents or reverses the development of diabetic complications (67,110).…”

Effect of rosuvastatin on high glucose-induced endoplasmic reticulum stress in human umbilical vein endothelial cells

“…Endothelial cells cultured in high glucose (HG) medium exhibit reduced nitric oxide production (Kemeny et al, 2013) and enhanced expression of inflammation-related genes (Safi et al, 2015). The mechanisms responsible may include accumulation of advanced glycation end products (Janket et al, 2008), increased reactive oxygen species (ROS) production (Cifarelli et al, 2011), activation of protein kinase C (PKC) signaling (Batchuluun et al, 2014), and endoplasmic reticulum (ER) stress (Schisano et al, 2012). We believe that this latter plays a crucial role in endothelial dysfunction, as it is capable of causing a series of pathological changes influencing the inflammatory response and apoptosis (Kim et al, 2007).…”