The effects of insulin and liraglutide on osteoprotegerin and vascular calcification in vitro and in patients with type 2 diabetes

Abstract

Objective: Vascular calcification (VC) is inhibited by the glycoprotein osteoprotegerin (OPG). It is unclear whether treatments for type 2 diabetes are capable of promoting or inhibiting VC. The present study examined the effects of insulin and liraglutide on i) the production of OPG and ii) the emergence of VC, both in vitro in human aortic smooth muscle cells (HASMCs) and in vivo in type 2 diabetes. Design/methods: HASMCs were exposed to insulin glargine or liraglutide, after which OPG production, alkaline phosphatase (ALP) activity and levels of Runx2, ALP and bone sialoprotein (BSP) mRNA were measured. A prospective, nonrandomised human subject study was also conducted, in which OPG levels and coronary artery calcification (CAC) were measured in a type 2 diabetes population before and 16 months after the commencement of either insulin or liraglutide treatment and in a control group that took oral hypoglycemics only. Results: Exposure to insulin glargine, but not liraglutide, was associated with significantly decreased OPG production (11 913G1409 pg/10 4 cells vs 282G13 pg/10 4 cells, control vs 10 nmol/l insulin, P!0.0001), increased ALP activity (0.82G0.06 IU/10 4 cells vs 2.40G0.16 IU/10 4 cells, control vs 10 nmol/l insulin, P!0.0001) and increased osteogenic gene expression byHASMCs. In the clinical study (nZ101), insulin treatment was associated with a significant reduction in OPG levels and, despite not achieving full statistical significance, a trend towards increased CAC in patients. Conclusion: Exogenous insulin down-regulated OPG in vitro and in vivo and promoted VC in vitro. Although neither insulin nor liraglutide significantly affected CAC in the present pilot study, these data support the establishment of randomised trials to investigate medications and VC in diabetes.

Serum osteoprotegerin level is positively associated with peripheral artery disease in patients with peritoneal dialysis

“…In DM patients, insulin was found to regulate the OPG production and vascular calcification in human aortic smooth muscle cells [29], and 24-week treatment of pioglitazone, which was a agonist of peroxisome proliferator-activated receptor gamma, could down-regulate serum levels of OPG as well as CRP [30]. In addition, treating patients with DM or CAD with atorvastatin or simvastatin appeared to lower serum OPG and high-sensitivity CRP as well as reduce arterial stiffness, which indicated beneficial effects of statin on vasculature through anti-inflammation [31,32].…”

Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells

“…Cell culture experiments were subsequently conducted in two formats: (i) Mono-culture experiments - HAECs and HASMCs were grown to confluency in 6-well dishes and separately treated for 72 h with either RANKL (0–25 ng/ml), TRAIL (0–5 ng/ml), or a combination of both (RANKL: 5 & 25 ng/ml + TRAIL: 5 ng/ml). This treatment period and dose range were previously established by Davenport et al [8,16] and Harper et al [7] and yielded robust responses. Post-treatment, cell lysates were harvested as previously described [17] in order to monitor activation of NF- κ B/ p52 (normalized to NF- κ B/ p100) and NF-κB/Phospho-p65 (normalized to NF- κ B/ p65) by Western blotting.…”

TRAIL attenuates RANKL-mediated osteoblastic signalling in vascular cell mono-culture and co-culture models

“…We investigated the dose-dependent effects of RANKL (0–25 ng/mL) on osteoblastic activity in both HAECs and HASMCs over 72 hr, a treatment period and dose range previously employed by Davenport et al . [ 12 , 28 ] and shown to yield robust responses. In parallel experiments designed to examine the protective effects of TRAIL towards RANKL-induced signalling, cells were grown to confluency in standard 6-well culture dishes and treated for 72 hr with RANKL (5 or 25 ng/mL), in the absence and presence of 5 ng/mL TRAIL.…”