Proinsulin C-peptide inhibits high glucose-induced migration and invasion of ovarian cancer cells.

Proinsulin C-peptide, a biologically active polypeptide released from pancreatic ß-cells, is known to prevent hyperglycemia-induced microvascular leakage; however, the role of C-peptide in migration and invasion of cancer cells is unknown. Here, we investigated high glucose-induced migration and invasion of ovarian cancer cells and the inhibitory effects of human C-peptide on metastatic cellular responses. In SKOV3 human ovarian cancer cells, high glucose conditions activated a vicious cycle of reactive oxygen species (ROS) generation and transglutaminase 2 (TGase2) activation through elevation of intracellular Ca2+ levels. TGase2 played a critical role in high glucose-induced ovarian cancer cell migration and invasion through ß-catenin disassembly. Human C-peptide inhibited high glucose-induced disassembly of adherens junctions and ovarian cancer cell migration and invasion through inhibition of ROS generation and TGase2 activation. The preventive effect of C-peptide on high glucose-induced ovarian cancer cell migration and invasion was further demonstrated in ID8 murine ovarian cancer cells. Our findings suggest that high glucose conditions induce the migration and invasion of ovarian cancer cells, and human C-peptide inhibits these metastatic responses by preventing ROS generation, TGase2 activation, and subsequent disassembly of adherens junctions.

Therapeutic effect of ultra-long-lasting human C-peptide delivery against hyperglycemia-induced neovascularization in diabetic retinopathy.

Rationale: Neovascularization is a hallmark of the late stages of diabetic retinopathy (DR) leading to blindness. The current anti-DR drugs have clinical disadvantages including short circulation half-lives and the need for frequent intraocular administration. New therapies with long-lasting drug release and minimal side effects are therefore needed. We explored a novel function and mechanism of a proinsulin C-peptide molecule with ultra-long-lasting delivery characteristics for the prevention of retinal neovascularization in proliferative diabetic retinopathy (PDR). Methods: We developed a strategy for ultra-long intraocular delivery of human C-peptide using an intravitreal depot of K9-C-peptide, a human C-peptide conjugated to a thermosensitive biopolymer, and investigated its inhibitory effect on hyperglycemia-induced retinal neovascularization using human retinal endothelial cells (HRECs) and PDR mice. Results: In HRECs, high glucose conditions induced oxidative stress and microvascular permeability, and K9-C-peptide suppressed those effects similarly to unconjugated human C-peptide. A single intravitreal injection of K9-C-peptide in mice resulted in the slow release of human C-peptide that maintained physiological levels of C-peptide in the intraocular space for at least 56 days without inducing retinal cytotoxicity. In PDR mice, intraocular K9-C-peptide attenuated diabetic retinal neovascularization by normalizing hyperglycemia-induced oxidative stress, vascular leakage, and inflammation and restoring blood-retinal barrier function and the balance between pro- and anti-angiogenic factors. Conclusions: K9-C-peptide provides ultra-long-lasting intraocular delivery of human C-peptide as an anti-angiogenic agent to attenuate retinal neovascularization in PDR.

Simultaneous attenuation of hyperglycemic memory-induced retinal, pulmonary, and glomerular dysfunctions by proinsulin C-peptide in diabetes.

BACKGROUND: Hyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin. METHODS: Insulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney. RESULTS: We found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage. CONCLUSIONS: Overall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.

Systemic C-peptide supplementation ameliorates retinal neurodegeneration by inhibiting VEGF-induced pathological events in diabetes.

Diabetic retinopathy (DR) is caused by retinal vascular dysfunction and neurodegeneration. Intraocular delivery of C-peptide has been shown to be beneficial against hyperglycemia-induced microvascular leakage in the retina of diabetes; however, the effect of C-peptide on diabetes-induced retinal neurodegeneration remains unknown. Moreover, extraocular C-peptide replacement therapy against DR to avoid various adverse effects caused by intravitreal injections has not been studied. Here, we demonstrate that systemic C-peptide supplementation using osmotic pumps or biopolymer-conjugated C-peptide hydrogels ameliorates neurodegeneration by inhibiting vascular endothelial growth factor-induced pathological events, but not hyperglycemia-induced vascular endothelial growth factor expression, in the retinas of diabetic mice. C-peptide inhibited hyperglycemia-induced activation of macroglial and microglial cells, downregulation of glutamate aspartate transporter 1 expression, neuronal apoptosis, and histopathological changes by a mechanism involving reactive oxygen species generation in the retinas of diabetic mice, but transglutaminase 2, which is involved in retinal vascular leakage, is not associated with these pathological events. Overall, our findings suggest that systemic C-peptide supplementation alleviates hyperglycemia-induced retinal neurodegeneration by inhibiting a pathological mechanism, involving reactive oxygen species, but not transglutaminase 2, in diabetes.

Dopamine ameliorates hyperglycemic memory-induced microvascular dysfunction in diabetic retinopathy.

Dopamine is a neurotransmitter that mediates visual function in the retina and diabetic retinopathy (DR) is the most common microvascular complication of diabetes and the leading cause of blindness; however, the role of dopamine in retinal vascular dysfunction in DR remains unclear. Here, we report a mechanism of hyperglycemic memory (HGM)-induced retinal microvascular dysfunction and the protective effect of dopamine against the HGM-induced retinal microvascular leakage and abnormalities. We found that HGM induced persistent oxidative stress, mitochondrial membrane potential collapse and fission, and adherens junction disassembly and subsequent vascular leakage after blood glucose normalization in the mouse retinas. These persistent hyperglycemic stresses were inhibited by dopamine treatment in human retinal endothelial cells and by intravitreal injection of levodopa in the retinas of HGM mice. Moreover, levodopa supplementation ameliorated HGM-induced pericyte degeneration, acellular capillary and pericyte ghost generation, and endothelial apoptosis in the mouse retinas. Our findings suggest that dopamine alleviates HGM-induced retinal microvascular leakage and abnormalities by inhibiting persistent oxidative stress and mitochondrial dysfunction.

C-peptide attenuates hyperglycemia-induced pulmonary fibrosis by inhibiting transglutaminase 2.

Proinsulin C-peptide has a protective effect against diabetic complications; however, its role in hyperglycemia-induced pulmonary fibrosis is unknown. In this study, we investigated the inhibitory effect of C-peptide on hyperglycemia-induced pulmonary fibrosis and the molecular mechanism of C-peptide action in the lungs of diabetic mice and in human pulmonary microvascular endothelial cells (HPMVECs). We found that, in the lungs of diabetic mice, C-peptide supplementation using osmotic pumps attenuated hyperglycemia-induced pulmonary fibrosis and expression of fibrosis-related proteins. In HPMVECs, C-peptide inhibited vascular endothelial growth factor-induced adherens junction disruption and endothelial cell permeability by inhibiting reactive oxygen species generation and transglutaminase (TGase) activation. In the lungs, C-peptide supplementation suppressed hyperglycemia-induced reactive oxygen species generation, TGase activation, and microvascular leakage. C-peptide inhibited hyperglycemia-induced inflammation and apoptosis, which are involved in the pathological process of pulmonary fibrosis. We also demonstrated the role of TGase2 in hyperglycemia-induced vascular leakage, inflammation, apoptosis, and pulmonary fibrosis in the lungs of diabetic TGase2-null (Tgm2-/-) mice. Furthermore, we demonstrated a long-term inhibitory effect of systemic delivery of C-peptide using K9-C-peptide hydrogels on hyperglycemia-induced fibrosis in diabetic lungs. Overall, our findings suggest that C-peptide alleviates hyperglycemia-induced pulmonary fibrosis by inhibiting TGase2-mediated microvascular leakage, inflammation, and apoptosis in diabetes.

Application of elastin-like biopolymer-conjugated C-peptide hydrogel for systemic long-term delivery against diabetic aortic dysfunction.

Due to their short half-lives, repeated administration of anti-hyperglycemic drugs can cause pain, discomfort, tissue damage, and infection in diabetic patients. Therefore, there is a need to develop long-term drug delivery systems to treat diabetes and its complications. C-peptide can prevent diabetic complications, including diabetic vasculopathy, but its clinical application is limited by its short half-life. Here, we developed K9-C-peptide (human C-peptide conjugated to an elastin-like biopolymer) and investigated its long-term influence on hyperglycemia-induced vascular dysfunction using an aortic endothelium model in diabetic mice. Using pharmacokinetics and in vivo imaging, we found that subcutaneously injected K9-C-peptide formed a hydrogel depot that slowly released human C-peptide into the blood circulation for 19 days. Administration of K9-C-peptide, human C-peptide, or K8 polypeptide had no effect on body weight or blood glucose levels. The slow release of C-peptide from K9-C-peptide hydrogels provided prolonged prevention of oxidative stress, inflammatory responses, and endothelial apoptosis in a hyperglycemia-induced vascular dysfunction model using the diabetic mouse aorta. Subcutaneous administration of unbound human C-peptide and K8 polypeptide were used as negative controls and had no effects. These results suggest that K9-C-peptide is suitable for the long-term delivery of human C-peptide for treating vascular dysfunction in diabetic patients.