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• W1582872317 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)-α 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-α 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 inflam-matory conditions and that anti-inflammatory approaches, such as anti-TNF-α strategies, improve diabetic wound healing. 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)-α 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-α 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 inflam-matory conditions and that anti-inflammatory approaches, such as anti-TNF-α strategies, improve diabetic wound healing. The functional connection between diabetes and foot ulceration was first recognized by the surgeon T.D. Pryce in 1887.1Pryce TD A case of perforating ulcers of both feet associated with diabetes and ataxic symptoms.Lancet. 1887; 2: 11-12Abstract Scopus (32) Google Scholar For the first time, Pryce claimed in an article published in Lancet that “diabetes itself may play an active part in the causation of perforating ulcers.”1Pryce TD A case of perforating ulcers of both feet associated with diabetes and ataxic symptoms.Lancet. 1887; 2: 11-12Abstract Scopus (32) Google Scholar Diabetic foot ulcers are skin lesions with a loss of epithelium that may extend into the dermis and may sometimes involve bone and muscle.2Boulton AJM The diabetic foot: from art to science. The 18th Camillo Golgi lecture.Diabetologia. 2004; 47: 1343-1353Crossref PubMed Scopus (284) Google Scholar, 3Reiber GE Ledoux WR Epidemiology of diabetic foot ulcers and amputations: evidence for prevention.in: Williams R Herman W Kinmonth AL Wareham NJ The Evidence Base for Diabetes Care. Wiley, Chichester, UK2002: 641-665Crossref Google Scholar It is now well established that ulcerations and subsequent amputation events of lower extremities represent serious complications of both types of diabetes mellitus and are associated with significant mortality.3Reiber GE Ledoux WR Epidemiology of diabetic foot ulcers and amputations: evidence for prevention.in: Williams R Herman W Kinmonth AL Wareham NJ The Evidence Base for Diabetes Care. Wiley, Chichester, UK2002: 641-665Crossref Google Scholar, 4Carrington AL Abbott CA Griffiths J Jackson N van Ross ERE Boulton AJM A foot care program for diabetic unilateral amputees.Diabetes Care. 2001; 24: 216-221Crossref PubMed Scopus (54) Google Scholar Thus, diabetic ulcers characterize an increasing clinical problem. The annual incidence of foot ulceration in the diabetic population is just over 2%,5Abbott CA Carrington AL Ashe H Bath S Every LC Griffiths J Hann AW Hussein A Jackson N Johnson KE Ryder CH Torkington R Van Ross ER Whalley AM Widdows P Williamson S Boulton AJ The North-West Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort.Diabetic Med. 2002; 20: 377-384Crossref Scopus (768) Google Scholar, 6Muller IS de Grauw WJ van Gerwen WH Bartelink ML van Den Hoogen HJ Rutten GE Foot ulceration and lower limb amputation in type 2 diabetic patients in Dutch primary health care.Diabetes Care. 2002; 25: 570-574Crossref PubMed Scopus (121) Google Scholar resulting in a lifetime risk of 15% for any diabetic patient to develop such a complication.7Jeffcoate WJ Harding KG Diabetic foot ulcers.Lancet. 2003; 361: 1545-1551Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 8Reiber GE Lipsky BA Gibbons GW The burden of diabetic foot ulcers.Am J Surg. 1998; 176: 5S-10SAbstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar Diabetic ulcers still have a poor prognosis, and the 3-year survival rates are between 50 and 59%, as assessed for Italy and Sweden, respectively.9Apelqvist J Larsson J Agardh CD Long-term prognosis for diabetic patients with foot ulcers.J Intern Med. 1993; 233: 485-491Crossref PubMed Scopus (375) Google Scholar, 10Faglia E Favales F Morabito A New ulceration, new major amputation, and survival rats in diabetic subjects hospitalized for foot ulceration from 1990 to 1993: a 6.5 year follow-up.Diabetes Care. 2001; 24: 78-83Crossref PubMed Scopus (160) Google Scholar By contrast, the efforts to identify novel pharmacological approaches to improve significantly severe diabetes-impaired healing conditions have failed. Only recombinant platelet-derived growth factor (becaplermin) is now available for treatment of foot ulcers.11Wieman TJ Smiell JM Su Y Efficacy and safety of topical gel formulation of recombinant human platelet-derived growth factor-BB (becaplermin) in patients with chronic neuropathic diabetic ulcers: a phase III randomized placebo-controlled double-blind study.Diabetes Care. 1998; 21: 822-827Crossref PubMed Scopus (552) Google Scholar Thus, Jeffcoate and Harding7Jeffcoate WJ Harding KG Diabetic foot ulcers.Lancet. 2003; 361: 1545-1551Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar focus the challenge for future research in their review article on diabetic foot ulceration by their demand that “investment is urgently needed for basic research into the pathophysiology of chronic wounds.” Here, we have used the obese/obese (ob/ob) mouse as a model system of diabetes-impaired wound healing. These mice are characterized by severe diabetes and obesity syndromes.12Coleman DL Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice.Diabetologia. 1978; 14: 141-148Crossref PubMed Scopus (1084) Google Scholar The diseased phenotype is mediated by a functional loss of the ob gene, which normally encodes a 16-kd cytokine named leptin.13Zhang Y Proenca R Maffei M Barone M Leopold L Friedman JM Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Crossref PubMed Scopus (11749) Google Scholar Severely impaired wound-healing conditions in ob/ob mice were strongly improved by administration of leptin, where leptin mediated wound re-epithelialization in a direct manner but attenuated chronic wound inflammation in an indirect manner.14Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (255) Google Scholar, 15Goren I Kämpfer H Podda M Pfeilschifter J Frank S Leptin and wound inflammation in diabetic ob/ob mice: differential regulation of neutrophil and macrophage influx and a potential role for the scab as a sink for inflammatory cells and mediators.Diabetes. 2003; 52: 2821-2832Crossref PubMed Scopus (87) Google Scholar In addition, systemic application of leptin to ob/ob mice also blunts both hyperglycemia and hyperinsulinemia and resolves the diabetic phenotype of the animals.14Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (255) Google Scholar, 15Goren I Kämpfer H Podda M Pfeilschifter J Frank S Leptin and wound inflammation in diabetic ob/ob mice: differential regulation of neutrophil and macrophage influx and a potential role for the scab as a sink for inflammatory cells and mediators.Diabetes. 2003; 52: 2821-2832Crossref PubMed Scopus (87) Google Scholar, 16Pelleymounter MA Cullen MJ Baker MB Hecht R Winters D Boone T Collins F Effects of the obese gene product on body weight regulation in ob/ob mice.Science. 1995; 269: 540-543Crossref PubMed Scopus (3869) Google Scholar It was reasonable to suggest that dysregulation and insensitivity of the insulin signaling machinery in resident skin cells might contribute to diabetes-impaired repair and that a leptin-driven adjustment of in-sulin sensitivity in skin tissue might be functionally connected to an improved healing in the animals. In line, skin keratinocytes have been shown to express the insulin receptor (InsR), which is functionally implicated in keratinocyte differentiation and glucose uptake.17Wertheimer E Spravchikov N Trebicz M Gartsbein M Accili D Avinoah I Nofeh-Moses S Sizyakov G Tennenbaum T The regulation of skin proliferation and differentiation in the IR null mouse: implications for skin complications of diabetes.Endocrinology. 2001; 142: 1234-1241Crossref PubMed Scopus (76) Google Scholar, 18Spravchikov N Sizyakov G Gartsbein M Accili D Tennenbaum T Wertheimer E Glucose effects on skin keratinocytes: implications for diabetes skin complications.Diabetes. 2001; 50: 1627-1635Crossref PubMed Scopus (164) Google Scholar There is increasing evidence for a functional link between insulin resistance, obesity, and diabetes. Initial studies demonstrated an increase in adipocyte-derived tumor necrosis factor (TNF)-α in obese rodents that was functionally connected to insulin resistance.19Hotamisligil GS Shargill NS Spiegelman BM Adipose expression of tumor necrosis factor-α: direct role in obesity-linked insulin resistance.Science. 1993; 259: 87-91Crossref PubMed Scopus (6144) Google Scholar Interestingly, plasma TNF-α levels were also dependent on adipose tissue mass in humans,20Kern PA Saghizadeh M Ong JM Bosch RJ Deem R Simsolo RB The expression of tumor necrosis factor in human adipose tissue: regulation by obesity, weight loss, and relationship to lipoprotein lipase.J Clin Invest. 1995; 95: 2111-2119Crossref PubMed Scopus (1169) Google Scholar, 21Mantzoros CS Moschos S Avramopoulos I Kaklamani V Liolios A Doulgerakis DE Griveas I Katsilambros N Flier JS Leptin concentrations in relation to body mass index and the tumor necrosis factor-α system in humans.J Clin Endocrinol Metab. 1997; 82: 3408-3413Crossref PubMed Scopus (289) Google Scholar and clinical studies confirmed that the presence of inflammatory mediators predicts the development of type 2 diabetes mellitus.22Schmidt MI Duncan BB Sharrett AR Lindberg G Savage PJ Offenbacher S Azambuja MI Tracy RP Heiss G Markers of inflammation and prediction of diabetes mellitus in adults (Atherosclerosis Risk in Communities Study): a cohort study.Lancet. 1999; 353: 1649-1652Abstract Full Text Full Text PDF PubMed Scopus (872) Google Scholar, 23Pradhan AD Manson JE Rifai N Buring JE Ridker PM C-reactive protein, interleukin 6, and risk of developing type 2 diabetes mellitus.JAMA. 2001; 286: 327-334Crossref PubMed Scopus (3399) Google Scholar, 24Barzilay JI Abraham L Heckbert SR Cushman M Kuller LH Resnick HE Tracy RP The relation of markers of inflammation to the development of glucose disorders in the elderly: the Cardiovascular Health Study.Diabetes. 2001; 50: 2384-2389Crossref PubMed Scopus (485) Google Scholar These observations suggest that obesity-associated inflammatory mediators such as TNF-α might contribute to insulin resistance in skin tissue. Using the leptin-deficient ob/ob mouse model, we investigated the insulin sensitivity of nonwounded and injured skin tissue under normal and diabetes-impaired conditions. Here, we provide evidence that disturbed insulin signaling pathways are associated with impaired repair in ob/ob mice and that TNF-α functionally interferes with insulin signaling and tissue regeneration at the wound site. Female C57BL/6J (wild-type) and C57BL/6J-ob/ob mice were obtained from The Jackson Laboratories (Bar Harbor, ME) and maintained under a 12-hour light/12-hour dark cycle at 22°C until they were 8 weeks of age. At this time, they were caged individually, monitored for body weight, and wounded as described below. Murine recombinant leptin (2 μg/g body weight) (Calbiochem, Bad Soden, Germany) and purified monoclonal anti-TNF-α antibody V1q25Echtenacher B Falk W Mannel DN Krammer PH Requirement of endogenous tumor necrosis factor/cachectin for recovery from experimental peritonitis.J Immunol. 1990; 145: 3762-3766PubMed Google Scholar (1 μg/g body weight) (Abcam Ltd., Cambridge, UK) were injected intraperitoneally in 0.5 ml of phosphate-buffered saline (PBS) for the indicated time periods. For local treatment, wounds of mice were covered with 1 μg of leptin in 20 μl of PBS twice a day (8:00 a.m. and 8:00 p.m.). Control mice were treated with PBS or an unspecific IgG (Santa Cruz, Heidelberg, Germany), respectively. Wounding of mice was performed as described previously.26Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Frank S the function of nitric oxide in wound repair: inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (196) Google Scholar, 27Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (214) Google Scholar Briefly, mice were anesthetized with a single intraperitoneal injection of ketamine (80 mg/kg body weight)/xylazine (10 mg/kg body weight). The hair on the back of each mouse was cut, and the back was sub-sequently wiped with 70% ethanol. Six full-thickness wounds (5 mm in diameter, 3 to 4 mm apart) were made on the back of each mouse by excising the skin and the underlying panniculus carnosus. The wounds were allowed to form a scab. Skin biopsy specimens were obtained from the animals 1, 3, 5, 7, and 13 days after injury. At each time point, an area that included the scab, the complete epithelial and dermal compartments of the wound margins, the granulation tissue, and parts of the adjacent muscle and subcutaneous fat tissue was excised from each individual wound. As a control, a similar amount of skin was taken from the backs of nonwounded mice. For each experimental time point, tissue from four wounds each from four animals (n = 16 wounds, RNA analysis) and from two wounds each from four animals (n = 8 wounds, protein analysis) were combined and used for RNA and protein preparation. Nonwounded back skin from four animals served as a control. All animal experiments were performed according to the guidelines and approval of the local Ethics Animal Review Board. RNA isolation and RNase protection assays were performed as described previously.27Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (214) Google Scholar, 28Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63183) Google Scholar The cDNA probes were cloned using reverse transcriptase-polymerase chain reaction. The probes corresponded to nucleotides 3718 to 4080 (for InsR, NM010568.1), nucleotides 1173 to 1391 (for protein tyrosine phosphatase [PTP]-1B, BC010191.1), nucleotides 4261 to 4621 (for InsR substrate [IRS]-1, NM010570.2), nucleotides 1969 to 2201 (for IRS-2, AF090738), nucleotides 1294 to 1592 (for glucose transporter [Glut]-4, BC014282.1), nucleotides 796 to 1063 (for cyclooxygenase [COX]-2, M64291), nucleotides 541 to 814 (for TNF-α, NM013693), nucleotides 1405 to 1649 (for TNF-α R1, p55, NM001065.2), nucleotides 1192 to 1475 (for TNF-α R2, p75, NM001066.2), and nucleotides 163 to 317 (for GAPDH, NM002046). Mice were wounded as described above. Animals were sacrificed at day 5 after injury. Complete wounds were isolated from the back, bisected, and frozen in tissue-freezing medium. Six-micrometer frozen sections were subsequently analyzed using immunohistochemistry as described previously.26Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Frank S the function of nitric oxide in wound repair: inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (196) Google Scholar Additionally, wounds were fixed in formalin and embedded in paraffin. Paraffin-fixed sections were stained with hematoxylin and eosin. Antiserum against the β-subunit of InsR (Santa Cruz) was used for immunodetection. Wound, muscle, and liver tissue and cell culture lysates were prepared as described previously.26Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Frank S the function of nitric oxide in wound repair: inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098Crossref PubMed Scopus (196) Google Scholar, 29Kämpfer H Kalina U Mühl H Pfeilschifter J Frank S Counterregulation of interleukin-18 mRNA and protein expression during cutaneous wound repair in mice.J Invest Dermatol. 1999; 113: 369-374Crossref PubMed Scopus (70) Google Scholar Fifty micrograms of total protein lysate was separated using sodium dodecyl sulfate (SDS)-gel electrophoresis, and specific proteins were detected using antisera directed against InsRβ, Glut-4 (Santa Cruz), PTP-1B, IRS-1, IRS-2 (Biomol, Hamburg, Germany), phospho-glycogen synthase kinase (GSK)-3α/β, phospho-glycogen synthase (GS), phospho-tyrosine (Cell Signaling, Frankfurt, Germany), or actin (Sigma, Deisenhofen, Germany). Five hundred micrograms of total wound protein lysate was incubated overnight with 2.5 μg of a monoclonal, biotinylated anti-phospho-tyrosine (4G10) antibody (Biomol). Next, 150 μg of streptavidin-coupled magnetic beads (MyOne; Dynal, Hamburg, Germany) was added for 2 hours. Magnetic beads were isolated and washed with PBS, and protein was eluted using Laemmli buffer. Quiescent, confluent human HaCaT keratinocytes were incubated in 35-mm wells with 1 ml of Krebs-Ringer solution (Sigma, Deisenhofen, Germany) in the presence or absence of insulin (0.1 and 1 μg/ml) for 20 minutes. Subsequently, 0.5 μCi of d-[2-H3]glucose (Amersham, Freiburg, Germany) was added per 35-mm well. Cells were harvested using 10% (w/v) SDS after different time points of incubation. Total wound lysate was analyzed for the presence of immunoreactive TNF-α by enzyme-linked immunosorbent assay (ELISA) using the Quantikine murine ELISA kit (R&D Systems, Wiesbaden, Germany). Blood glucose levels were determined using the Accutrend sensor (Roche Biochemicals, Mannheim, Germany). Serum insulin and leptin were analyzed by ELISA (Crystal Chemicals, Chicago, IL) as described by the manufacturer. Viability of cultured keratinocytes was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay following a published protocol30Mosmann T Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J Immunol Methods. 1983; 65: 55-63Crossref PubMed Scopus (46481) Google Scholar and the lactate dehydrogenase assay. The lactate dehydrogenase assay was performed according to the instructions of the manufacturer (Roche Biochemicals, Mannheim, Germany). Data are shown as means ± SD. Data analysis was performed using the unpaired Student's t-test with raw data. Statistical comparison between more than two groups was performed by analysis of variance (Dunnett's method). In this study, we investigated the availability of insulin signaling pathways in skin of normal and diabetes-impaired wound healing. The ob/ob mouse is characterized by a severe type 2 diabetes mellitus12Coleman DL Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice.Diabetologia. 1978; 14: 141-148Crossref PubMed Scopus (1084) Google Scholar and suffers from disturbed wound-healing conditions.14Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (255) Google Scholar, 15Goren I Kämpfer H Podda M Pfeilschifter J Frank S Leptin and wound inflammation in diabetic ob/ob mice: differential regulation of neutrophil and macrophage influx and a potential role for the scab as a sink for inflammatory cells and mediators.Diabetes. 2003; 52: 2821-2832Crossref PubMed Scopus (87) Google Scholar, 31Ring BD Scully S Corrine CR Baker MB Cullen MJ Pelleymounter MA Danilenko DM Systemically and topically administered leptin both accelerate wound healing in diabetic ob/ob mice.Endocrinology. 2000; 141: 446-449Crossref PubMed Scopus (107) Google Scholar We found that expression of InsR (Figure 1A) and Glut-4 (Figure 1B) mRNA was significantly reduced in impaired wounds of diabetic ob/ob mice when compared with acutely healing wounds of C57BL/6 mice. By contrast, mRNA expression levels did not markedly change during healing in both groups for PTP-1B, IRS-1, and IRS-2 (data not shown). However, protein expression revealed marked differences in the availability and activation of insulin-sensitive components in skin tissue of control and diseased mice. In clear contrast to healthy mice, the constitutively expressed InsR completely diminished on injury in diabetes-impaired wound tissue (Figure 2A). Notably, InsR and PTP-1B expression during normal healing paralleled a reduced amount of protein during the acute repair process (Figure 2, A and B, top panels, 1 day wound to 7 days wound). However, PTP-1B was barely detectable in ob/ob mice (Figure 2B, bottom panel). PTP-1B represents a central negative regulator of insulin action32Johnson TO Ermolieff J Jirousek MR Protein tyrosine phosphatase 1B inhibitors for diabetes.Nat Rev Drug Discov. 2002; 1: 696-709Crossref PubMed Scopus (563) Google Scholar that is inactivated by tyrosine phosphorylation.33Tao J Malbon CC Wang HY Insulin stimulates tyrosine phosphorylation and inactivation of protein-tyrosine phosphatase 1B in vivo.J Biol Chem. 2001; 276: 29520-29525Crossref PubMed Scopus (65) Google Scholar Interestingly, we observed an increase of tyrosine phosphorylated and thus inactive PTP-1B (Figure 2C, top panel, 1 day wd to 7 days wd) that paralleled InsR down-regulation (Figure 2A) during acute healing in control mice. Nevertheless, the small amounts of PTP-1B in wounds of ob/ob mice were most likely inactive, because tyrosine phosphorylation of PTP-1B increased during repair (Figure 2C, bottom panel). We also found the expression of InsR adaptor molecules IRS-1 (Figure 2D) and IRS-2 (Figure 2E) to be markedly reduced in skin and wounds of diabetic ob/ob mice. Finally, we investigated possible consequences of altered InsR, PTP-1B, or IRS-1 and −2 availability during diabetes-impaired repair. To this end, we determined activation of the key down-stream molecules GSK3α and -β and GS in normal and wounded skin. Insulin causes inactivation of GSK3 as a result of serine phosphorylation of the kinase, finally leading to serine dephosphorylation and functional activation of GS.34Doble BW Woodgett JR GSK-3: tricks of the trade for a multi-tasking kinase.J Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1764) Google Scholar We observed increasing amounts of phosphorylated GSK3α (Ser21) and GSK3β (Ser9) during normal repair, which were nearly completely absent in ob/ob mice (Figure 2F). However, although wound GSK3α/β activity was most likely blunted by serine phosphorylation, we recognized a marked phosphorylation (Ser641) of GS during the complete healing period in healthy mice, which did not persist in diseased animals (Figure 2G). Moreover, Glut-4 was constitutively expressed during normal repair but was strongly down-regulated in diabetes-impaired wound conditions (Figure 2H).Figure 2Key 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunohistochemistry revealed the InsR to be expressed in proliferating keratinocytes located at the wound margins and within the granulation tissue in 5-day wound tissue of healthy mice (Figure 3A), indicating that both epidermal and mesenchymal wound cells were potentially sensitive toward insulin. Notably, we could barely detect InsR-specific signals in wound sections isolated from diabetic ob/ob mice (Figure 3B), which were characterized by clearly reduced hyperproliferative epithelia located at the margins of the wound.14Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (255) Google Scholar It is well established that systemic treatment of leptin-deficient ob/ob mice with recombinant leptin results in resolution of both the diabetic and impaired healing phenotype.14Frank S Stallmeyer B Kämpfer H Kolb N Pfeilschifter J Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (255) Google Scholar, 16Pelleymounter MA Cullen MJ Baker MB Hecht R Winters D Boone T Collins F Effects of the obese gene product on body weight regulation in ob/ob mice.Science. 1995; 269: 540-543Crossref PubMed Scopus (3869) Google Scholar Thus, ob/ob mice were injected intraperitoneally with recombinant leptin (2 μg/g body weight, once a day) for 13 days. As shown in Figure 4A, we found high serum leptin levels 3 hours after injection. The diabetic phenotype of leptin-injected ob/ob mice was resolved, because hyperinsulinemia and blood glucose were rapidly adjusted to normal. Moreover, after 13 days of leptin treatment, mice revealed a significant loss of body weight (Figure 4A) and an improved healing, as assessed by the loss of scabs after wound re-epithelialization and reduction of wound areas (Figure 4, B and C). Histological analysis of 13-day wound tissue convincingly demonstrated the potency of systemically administered leptin to improve wound re-epithelialization in ob/ob mice (Figure 4D). Late wound areas from leptin-treated mice were characterized by a robust formation of a multilayered and organized neo-epidermis and neo-dermis, which completely covered the site of injury (Figure 4D, left panels). By contrast, wounds of PBS-treated mice revealed only small and reduced neo-epithelia at the wound margins and completely failed to cover the site of injury with a well-developed granulation tissue and neo-epithelium. At this stage of impaired healing, wound coverage was represented not by cells but by a robust scab (Figure 4D, right panels). Next, we investigated the presence and activation of key insulin signaling molecules in late chronic and improved wounds (day 13 after wounding) isolated from PBS- or leptin-treated ob/ob mice. We could not detect significant changes in total InsR, PTP-1B, IRS-1, and Glut-4 mRNA expression in improved and diabetes-impaired wounds, but IR" @default.
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• W1582872317 date "2006-03-01" @default.
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• W1582872317 title "Severely Impaired Insulin Signaling in Chronic Wounds of Diabetic ob/ob Mice" @default.
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• W1582872317 doi "https://doi.org/10.2353/ajpath.2006.050293" @default.
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