Severely impaired insulin signaling in chronic wounds of diabetic ob/ob mice: a potential role of tumor necrosis factor-alpha

Abstract

Wound-healing disorders are major complications of diabetes mellitus. Here, we investigated insulin-mediated signaling in nonwounded skin and in cutaneous tissue regeneration of healthy C57BL/6 and diabetes-impaired leptin-deficient obese/obese (ob/ob) mice. The insulin receptor (InsR) was abundantly expressed in wound margins and granulation tissue during acute healing in healthy mice. Remarkably, active signaling from the InsR, as assessed by phosphorylation of downstream targets such as protein tyrosine phosphatase-1B, glycogen synthase (GS), and GS kinase, was nearly absent in nonwounded and acutely healing skin from ob/ob mice. Systemic leptin administration to ob/ob mice reverted the diabetic phenotype and improved tissue regeneration as well as the impaired expression of InsR, insulin receptor substrate-1 and insulin receptor substrate-2, and downstream signaling (phosphorylation of GS kinase and GS) in late wounds and nonwounded skin of ob/ob mice. Importantly, tumor necrosis factor (TNF)-alpha was a mediator of insulin resistance in keratinocytes in vitro and in ob/ob wound tissue in vivo. Systemic administration of a monoclonal anti-TNF-alpha antibody (V1q) in wounded ob/ob mice attenuated wound inflammation, improved re-epithelialization, and restored InsR expression and signaling in wound tissue of ob/ob mice. These data suggest that InsR signaling in diabetes-impaired wounds is sensitive to inflammatory conditions and that anti-inflammatory approaches, such as anti-TNF-alpha strategies, improve diabetic wound healing.

Figure 1

Expression of insulin signaling molecules in skin repair. Regulation of InsR (A) and Glut-4 (B) mRNA expression in nonwounded back skin (ctrl) and wound tissue isolated from wild-type (C57BL/6) and ob/ob mice. The time after injury is indicated. Each single experimental time point represents 16 wounds (n = 16) isolated from four individual mice of two experimental series. Mean expression levels during repair are shown in the right panels. **, P < 0.01; *, P < 0.05. Bars indicate the mean ± SD over the healing period (days 1 to 13) from 80 wounds from 20 animals.

Figure 2

Key proteins of the insulin signaling cascade during normal and diabetes-impaired wound (wd) healing. Immunoblots showing InsR (A), PTP-1B (B), tyrosine-phosphorylated (Y-P) PTP-1B (C), IRS-1 (D), IRS-2 (E), phosphorylated (S21, S9) GSK3α/β (F), phosphorylated (S641) GS (G), and Glut-4 (H) in nonwounded (ctrl skin) and wounded skin in C57BL/6 and ob/ob mice as indicated. A control for equal loading (Ponceau S staining) is shown in I. J: Integrity of protein lysates is again controlled by immunodetection of actin. The time after injury is indicated. Each time point depicts eight wounds (n = 8) from four individual mice (n = 4). Liver and muscle tissue was used as control tissue to prove specificity of antibodies.

Figure 3

Induction and localization of the InsR at the wound site. Immunohistochemical localization of InsR protein in 5-day wounds of healthy C57BL/6 (A) and diabetic ob/ob mice (B). Particularly strong signals for InsR expression are indicated by arrows. he, hyperproliferative epithelium; gt, granulation tissue; sc, scab.

Figure 4

Administered leptin is biologically active. A: Blood leptin, insulin, and glucose levels 3 hours after systemic application of recombinant leptin. The ob/ob mice were treated with leptin for 13 days, after which the body weight of the animals was monitored. B: Presence of scab-covered wounds and wound area after 13-day treatment with PBS or leptin. **, P < 0.01 as compared with PBS-treated animals. Bars indicate the means ± SD from nine individual animals (n = 9). C: Photographs of 13-day wounds in PBS- or leptin-treated ob/ob mice. D: Representative histological analysis of a 13-day wound tissue of a leptin-treated (left panels) or PBS-treated (right panels) ob/ob mouse. Top panels show the wound margin area (site of initial injury is marked by an asterisk); bottom panels show the middle of the wound. gt, granulation tissue; nd, neo-dermis; ne, neo-epidermis; sc, scab.

Figure 5

Disturbed key components of insulin signaling are adjusted by systemic leptin administration in wounds of diabetic ob/ob mice. Immunoblots showing InsR, phospho-InsR, PTP-1B, and phospho-PTP-1B (A); IRS-1 and IRS-2 (B); phospho-GSK3α/β and phospho-GS (C); and Glut-4 (D) in 13-day wounds of PBS- and leptin-treated ob/ob mice as indicated. Numbers indicate individual mice. Each time point depicts eight wounds (n = 8) from four individual mice (n = 4). Liver and muscle tissue and lysates from IRS-1- and IRS-2-overexpressing cells were used as controls to demonstrate specificity of antibodies.

Figure 6

Topical treatment of wounds (wd) with leptin did not improve InsR expression. A: Blood insulin and glucose levels 3 hours after topical application of recombinant leptin (1 μg/wound). After 13 days of leptin treatment, the body weight of the animals was monitored. Bars indicate the means ± SD from four individual animals (n = 4). B: Immunoblots showing InsR expression in nonwounded skin (ctrl skin) and wounds of topically treated ob/ob mice as indicated. Each time point depicts eight wounds (n = 8) from four individual mice (n = 4). Thirteen-day wound tissue from leptin-injected mice and muscle tissue served as positive controls.

Figure 7

Leptin adjusts key molecules of insulin signaling in nonwounded diabetic skin tissue. ob/ob mice were systemically treated with PBS or leptin (Lep) for 7 days. Immunoblots showing InsR (A), IRS-1 (B), PTP-1B (C), phospho-GSK3α/β (D), phospho-GS (E), and Glut-4 (F) in nonwounded skin of PBS- and leptin-treated ob/ob mice as indicated. Numbers indicate individual mice. Skin of C57BL/6 mice, or liver and muscle tissue were used as controls.

Figure 8

The role of chronic insulin and TNF-α exposure for insulin actions in cultured keratinocytes. Serum-starved HaCaT keratinocytes were stimulated for glucose uptake (x-fold compared with control) in the presence or absence of chronic insulin (A) or TNF-α (B) pretreatment as indicated. **, P < 0.01; *, P < 0.05 as compared with controls. Bars indicate the means ± SD obtained from four independent cell culture experiments (n = 4). Keratinocyte viability as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (C) or lactate dehydrogenase (D) assay in the presence or absence of a 72-hour TNF-α stimulation as indicated. E: Immunoblot demonstrating phospho-GSK3β on insulin stimulation in the presence or absence of TNF-α pretreatment. F: RNase protection assay demonstrating mRNA expression of TNF-α receptor 1 (TNFR1, p55) and TNF-α receptor 2 (TNFR2, p75) in starved HaCaT keratinocytes on TNF-α (2 nmol/L) stimulation as indicated. Hybridization against tRNA was used as a negative control.

Figure 9

Systemic neutralization of TNF-α strongly improves diabetes-impaired wound healing. A: Quantification of TNF-α mRNA (RNase protection assay; left panel) and protein (ELISA; right panel) expression in 13-day wounds from PBS- and leptin (Lep)-treated ob/ob mice. **, P < 0.01 as indicated by the brackets. Bars indicate the means ± SD obtained from 18 wounds (n = 18) isolated from nine animals (n = 9). B: COX-2 mRNA expression in 13-day wound tissue from ob/ob mice that have been treated with PBS, leptin, unspecific control antibody (IgG), or a monoclonal anti-TNF-α antibody (V1q) as indicated. *, P < 0.05 as indicated by brackets. Bars indicate the means ± SD obtained from wounds (n = 16) isolated from four different animals (n = 4). C: Immunoblot showing the expression of InsR and phospho-GSK3α in 13-day wounds from IgG-, leptin-, or anti-TNF-α antibody (V1q)-injected ob/ob mice as indicated. Ponceau S staining of the blot is shown as a loading control. D: Photographs of 13-day wounds in IgG-, leptin-, and anti-TNF-α (V1q)-treated ob/ob mice. E: Presence of scab-covered wounds and wound area of 13-day wounds in IgG-, leptin-, and anti-TNF-α (V1q)-treated ob/ob mice. **, P < 0.01 as indicated by the brackets. Bars indicate the means ± SD from 18 wounds (n = 18) from three individual animals (n = 3). F: Histological analyses of 13-day wound tissue isolated from three (n = 3) individual anti-TNF-α (V1q)- or IgG-treated ob/ob mice as indicated. Formalin-fixed, paraffin-embedded 6-μm sections were counterstained using hematoxylin and eosin. at, adipose tissue; gt, granulation tissue; gt*, atrophied gt; ne, neo-epidermis, sc, scab.