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.

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.