Topical Insulin Accelerates Wound Healing in Diabetes by Enhancing the AKT and ERK Pathways: A Double-Blind Placebo-Controlled Clinical Trial

Background: Wound healing is impaired in diabetes mellitus, but the mechanisms involved in this process are virtually unknown. Proteins belonging to the insulin signaling pathway respond to insulin in the skin of rats. Objective: The purpose of this study was to investigate the regulation of the insulin signaling pathway in wound healing and skin repair of normal and diabetic rats, and, in parallel, the effect of a topical insulin cream on wound healing and on the activation of this pathway. Research Design and Methods: We investigated insulin signaling by immunoblotting during wound healing of control and diabetic animals with or without topical insulin. Diabetic patients with ulcers were randomized to receive topical insulin or placebo in a prospective, double-blind and placebo-controlled, randomized clinical trial (NCT 01295177) of wound healing. Results and Conclusions: Expression of IR, IRS-1, IRS-2, SHC, ERK, and AKT are increased in the tissue of healing wounds compared to intact skin, suggesting that the insulin signaling pathway may have an important role in this process. These pathways were attenuated in the wounded skin of diabetic rats, in parallel with an increase in the time of complete wound healing. Upon topical application of insulin cream, the wound healing time of diabetic animals was normalized, followed by a reversal of defective insulin signal transduction. In addition, the treatment also increased expression of other proteins, such as eNOS (also in bone marrow), VEGF, and SDF-1 alpha in wounded skin. In diabetic patients, topical insulin cream markedly improved wound healing, representing an attractive and cost-free method for treating this devastating complication of diabetes.Sao Paulo Research Foundation (FAPESP)Sao Paulo Research Foundation (FAPESP)National Institute of Science and Technology (INCT)National Institute of Science and Technology (INCT)National Council for Scientific and Technological Development (CNPq)National Council for Scientific and Technological Development (CNPq

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Effect of insulin on cellular and molecular mechanisms of wound healing in diabetes.

<p>Insulin induces activation of IR/SHC/ERK and IR/IRS/PI3K/AKT pathways in wound healing, which are canonical insulin signaling pathways. On the upper right-hand side, AKT is shown to increase VEGF (probably from macrophages, fibroblasts and epithelial cells) that will induce the phosphorylation and activation of eNOS in bone marrow, with consequent mobilization of EPCs to the circulation. SDF1α induces the homing of these EPC at the injury site, where they participate in neovasculogenesis. Insulin cream increased VEGF and SDF1α tissue expression in wound healing, and also increased eNOS phosphorylation in the bone marrow of an animal model of diabetes.</p

Time-course of IRS-1 and AKT expression following skin wounding in control (A, B) and diabetic animals (C, D).

<p>Tissue protein levels in the intact skin of control (CC) and intact skin of diabetic (DD) rats and in the wounded skin of control (WC) and wounded skin of diabetic animals (WD). Skin and wound extracts from control and diabetic rats were prepared, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036974#s2" target="_blank"><i>Materials and Methods</i></a>, four days after the wound incision. Tissue extracts were immunoblotted with (E) anti-IR antibody, (F) anti-IRS-1 antibody, (G) anti-IRS-2 antibody, (H) anti-SHC antibody, (I) anti-AKT antibody, and (J) anti-ERK1/2 antibody. Equal protein loading was confirmed by reblotting the membranes with anti-β-actin. Data were compared by ANOVA and Bonferroni post-test, and represented by the mean and standard deviation for each group of scanning densitometry of six different animals per group. *P<0.05 between groups.</p

Days to achieve complete healing in wounded control rats (WC), wounded control rats that received the insulin cream (WCI), wounded diabetic rats (WD), and wounded diabetic rats that received insulin cream (WDI).

<p>(A) Wound area was quantified every day and expressed as the percentage of the original wound area. (B) Wound extracts were prepared as described in<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036974#s2" target="_blank"><i>Materials and Methods</i></a> four days after the wound incision and were immunoblotted with (C) anti-IRS-1, (D) anti-SHC, (E) anti-phospho-AKT, (F) anti-phospho-ERK1/2, (G) anti-phospho-GSK3, and (H) anti-phospho-eNOS antibodies. To determine the protein levels of AKT, ERK1/2, GSK3, and eNOS, the membranes were stripped and reprobed with anti-AKT, -ERK1/2, -GSK3 and -eNOS. Equal protein loading was confirmed by reblotting the membranes with anti-β-actin. Data were compared by ANOVA and Bonferroni post-test, and represented by the mean and standard deviation for each group of scanning densitometry of six different animals per group, and the bar graphs represent the ratio of phosphorylation/protein. *p<0.05 between groups; **p<0.05 <i>vs.</i> WCI.</p

Effect of the inhibitors of PI3K and of ERK. Effect of inhibitors of PI3K (LY294002) and/or ERK (PD98059) on wound healing on day six after beginning use of the topical cream, represented as a percentage of change in wound area in wounded diabetic rats (WD), wounded diabetic rats treated with LY294002 (WD+Ly), wounded diabetic rats treated with PD98059 (WD+PD), wounded diabetic rats treated with insulin (WDI), wounded diabetic rats treated with insulin and Ly (WD+Ly), wounded diabetic rats treated with insulin and PD (WD+PD), and wounded diabetic rats treated with insulin, Ly and PD (WDI+Ly+PD).

<p>(A) AKT phosphorylation in wound healing. (B) ERK1/2 phosphorylation in wound healing. (C) VEGF-1 protein expression in wound healing. (D) SDF-1α protein expression in wound healing. (E) eNOS phosphorylation and eNOS protein expression in wound healing. (F) Equal protein loading was confirmed by reblotting the membranes with anti-β-actin. Data were compared by ANOVA and Bonferroni post-test, and represented by the mean and standard deviation for each group of scanning densitometry of six different animals per group. *P<0.05 between groups; **P<0.05 <i>vs.</i> WD <i>vs.</i> WD+Ly <i>vs.</i> WD+PD; ***P<0.05 <i>vs.</i> WDI.</p

Clinical and laboratory characteristics of diabetic patients, treated with placebo (P) or insulin cream (I).

<p>There were no significant differences between the groups, according to a paired t-test.</p

Plasma glucose levels of 10 diabetic and 10 control animals that received the cream with insulin or with placebo.

<p>The values are represented as the mean ± SEM of 10 experiments. WC: wounded control rats; WCI: wounded control rats treated with insulin cream; WD: diabetic animals treated with placebo cream; WDI: diabetic rats treated with insulin cream. P<0.05 between control and diabetic rats.</p

Macroscopic wound closure in diabetic rats treated with different concentrations of insulin cream.

<p>Representative wound area values during the time-course of 16 days. Values are expressed as mean ± SEM of at least 10 animals per group. * p<0.05 comparing 0.5 U and 1.0 U <i>vs.</i> 0.0 U, 0.25 U, and 0.1 U of insulin/100 g of cream. (A) Photos showing the time-course of wound healing in rats 0, 4, and 8 days after they received the insulin cream. (B) Morphology of the wounds after four days (a–b) and eight days (c–d) in rats treated with WD cream (left column - a, c) or rats treated with WDI cream (right column - b, c). Arrowheads in c show the extremities of the unclosed wound, while d shows complete closure of the epithelium. EP = epidermis, DE = dermis, SC = scab. Magnification bar in D = 50 µm.</p