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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 2 847-851
Copyright © 2004 by The Endocrine Society

Insulin-Degrading Activity in Wound Fluid

William C. Duckworth, Janet Fawcett, Sreedevi Reddy and Jeffrey C. Page

Carl T. Hayden Veterans Affairs Medical Center, Phoenix, Arizona 85012

Address all correspondence and requests for reprints to: William C. Duckworth, M.D., Carl T. Hayden Veterans Affairs Medical Center (CS111E), 650 East Indian School Road, Phoenix, Arizona 85012


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Patients with diabetes are at great risk of developing lower extremity ulcers. The management of diabetic foot ulcers typically includes early recognition and appropriate clinical care. Recent advances in wound treatment include topical growth factor therapy, which has been successful in diabetic wounds. Growth factors are decreased in wound fluid; this may be due to decreased supply, increased binding, or increased degradation of the naturally occurring growth factors. This study investigates the activity of the insulin-degrading enzyme in wound fluid. Wound fluid was obtained from patients with (n = 17) and without (n = 4) diabetes. Insulin degradation was assayed by incubating [125I]insulin with wound fluid and precipitation in trichloroacetic acid. Fluid from nondiabetics degraded 2.22 ± 0.73%, whereas diabetic fluid degraded significantly more (6.13 ± 1.48%; P < 0.05). In patients with diabetes, the degradation of insulin by wound fluid correlated with glucose control (hemoglobin A1c; r2 = 0.5353; P < 0.001), and patients with worse outcomes (i.e. amputation) had higher wound fluid insulin degradation. The biochemical characteristics of insulin degradation in the wound fluid were consistent with the characteristics of insulin-degrading enzyme. These data suggest that glucose control is a critical factor in wound healing, but a reduction in the insulin-degrading activity in the wound fluid is also a potential therapeutic target.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
LOWER EXTREMITY ULCERS are a major cause of morbidity and mortality in patients with diabetes. The primary factors in the development of these lesions are vascular insufficiency and peripheral neuropathy. Decreased tissue blood flow and insensitivity to pain result in tissue damage and poor healing of what might otherwise be minor lesions. Approximately 20% of diabetic patients with foot ulcers will primarily have inadequate arterial blood flow, approximately 50% will primarily have neuropathy, and approximately 30% will have both conditions (1, 2). A recent report from the United Kingdom showed that 7.4% of all patients with diabetes had a history of current or previous foot ulcer (3). The lifetime risk is 15%. Similar data have been reported from other countries. Management of diabetic foot ulcers typically includes early recognition, careful monitoring, improvement in glucose control, the correction of any underlying etiological factors, the establishment of adequate blood flow, the elimination of infection, the assurance of adequate nutrition, wound bed preparation (debridement of necrotic tissue, reduction of bacterial burden, and the use of a moist wound dressing), and the prevention of further trauma to the wound (4). Recent technological advances offer new interactive therapies that may serve to accelerate wound healing (5). These include topical growth factors, bioengineered tissue, negative pressure therapy, and many others.

Growth factor therapy has shown considerable promise in wound therapy in patients with diabetes (4, 5, 6). Topically applied growth factors have significantly accelerated wound repair in diabetic wounds. One possible reason for their success is the relative deficiency of growth factors in chronic wound fluid due to decreased supply, increased binding, or increased degradation of the naturally occurring growth factors (5).

The present study was performed to examine the level of the insulin-degrading enzyme (IDE) in diabetic wound fluid and relate this to glucose control [hemoglobin A1c (HbA1c) levels]. Similar to other proteases in chronic wound fluid, this enzyme degrades a number of growth factors in addition to insulin and has been shown to be present in the fluid from experimental wounds in animals (7). An excess of this enzyme could contribute to reduced levels of growth factors in wound fluid.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patient selection

All patients with lower extremity ulcers seen at this hospital with clinical indications for the use of Wound V.A.C. (Vacuum-Assisted Closure) therapy were invited to participate in this study. This study was approved by the institutional review board at the Carl T. Hayden Veterans Affairs Medical Center, and all subjects who consented were included. Infected wounds were excluded from the study.

Procedure

Negative pressure therapy using V.A.C. (Kinetic Concepts, Inc., San Antonio, TX) aids in the treatment of a variety of wounds with the aim of increasing the rate of wound healing, increasing patient comfort, decreasing the length of hospitalization, and decreasing the cost of care (8, 9, 10, 11, 12, 13). The technique involves the application of subatmospheric pressure (suction) to the wound bed through a sterile, polyurethane or polyvinyl alcohol, open-pore, foam sponge trimmed to fit within the wound bed. The sponge is covered with a thin transparent film to create a seal. One end of a suction tube is inserted into or attached to the foam. The opposite end of the tubing is attached to an adjustable vacuum source. The fluid collects in a canister inside the vacuum housing. Accumulated wound fluid was removed in this study no more than every 8 h. Thus, the Wound V.A.C. canister provided a ready source of wound fluid. Wound V.A.C. fluid was removed from the container, immediately placed on ice, and delivered to the laboratory. The fluid was separated from any cells or debris by centrifugation and either assayed immediately or aliquots were frozen at -80 C for later analysis.

Materials

[125I]A14-insulin was a gift from Eli Lilly & Co. (Indianapolis, IN). N-Ethylmaleimide (NEM) and bacitracin were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All other chemicals were at least reagent grade.

Insulin degradation assay

Wound fluid (150 µl) was added to 750 µl 100 mM Tris containing 1% BSA and placed in an ice bath for 30 min. [125I]insulin (10,000 cpm in 100 µl) was added, the samples were vortexed and incubated for 30, 60, and 90 min at 37 C. At the end of the incubation, 10% (final concentration) trichloroacetic acid (TCA) was added. Degradation was taken as the percentage of TCA-soluble radioactivity (14, 15). Inhibitors were added just before incubation in the ice bath. Inhibitors were added as 100x stocks to achieve the following final concentrations: aprotinin, 0.3 µM; EDTA, 10 mM; pepstatin A, 5 µM; bacitracin, 1 mM; and NEM, 1 mM. Inhibitor experiments were carried out for 90 min at 37 C.

Statistical analysis

Data were analyzed by unpaired t test or linear regression as appropriate using GraphPad PRISM (version 3.02). P < 0.05 was taken as significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Fluid from 21 patients was examined. Patients with diabetes comprised the great majority, and in fact, one of the "nondiabetic" subjects was found to have historical data sufficient for the diagnosis of diabetes and has been included in the diabetic group after chart review. This represents the usual distribution of wounds appropriate for Wound V.A.C. therapy, which are far more common in diabetes. The clinical characteristics of the subjects studied are shown in Table 1Go. Some of the subjects had more than one sample analyzed. Table 2Go shows the characteristics of the 17 patients with diabetes.


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  		TABLE 1. Clinical characteristics of the patients studied

 

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  		TABLE 2. Clinical characteristics of diabetic subjects

 
Figure 1AGo shows the time course of insulin degradation by wound fluid samples in patients with and without diabetes. Diabetic wound fluid had an increased amount of degradation (P < 0.05 at 90 min). Figure 1BGo shows the individual values for insulin degradation for the nondiabetic and diabetic subjects. At 30 min, all nondiabetic subjects had less than 2% degradation, whereas six of the 17 diabetic subjects had more than 2%. At 90 min, 10 of the 17 diabetic subjects had more degradation than any of the nondiabetic subjects.



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  		FIG. 1. Time course of insulin degradation by wound fluid from patients with (n = 17) and without (n = 4) diabetes (A; mean ± SEM). Individual sample data are shown in B. Insulin degradation was measured as described in Subjects and Methods, with the percentage of TCA soluble taken as degraded material. *, P < 0.05.

 
Figure 2Go shows that the amount of insulin degradation correlates with the HbA1c levels, i.e. the higher the HbA1c value, the greater the insulin degradation (r2 = 0.5353; P < 0.001). The clinical outcome of the wound (i.e. whether the wound healed or amputation was required) was also correlated to insulin-degrading activity and HbA1c levels (Fig. 3Go). Patients who required amputation had significantly higher insulin-degrading activities relative to HbA1c levels than patients whose ulcers healed.



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  		FIG. 2. Correlation of insulin degradation in wound fluid from patients with diabetes (n = 17) with HbA1c levels. Patients with higher HbA1c levels had greater insulin degradation in wound fluid (r2 = 0.5353; P < 0.001).

 


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  		FIG. 3. Correlation of insulin degradation in wound fluid from patients with diabetes (n = 15) with HbA1c levels: separation of wound outcomes (n = 7 for patients with healed wounds; n = 7 for patients requiring amputation). Patients that had wounds that remained open are not shown in this figure because there were too few to analyze (n = 3). Patients who required amputation had significantly higher insulin degradation values than those who healed (for healed: r2 = 0.8478; P < 0.005; for amputated: r2 = 0.8649; P < 0.005).

 
We investigated the characteristics of the wound fluid insulin-degrading activity with the use of inhibitors (Fig. 4Go). Bacitracin, NEM, and EDTA all significantly inhibited this activity. Pepstatin A and aprotinin had no effect. These findings are consistent with the characteristics of IDE (16).



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  		FIG. 4. Effect of inhibitors of IDE (bacitracin, NEM, and EDTA) and other proteases (pepstatin A and aprotinin) on insulin degradation in wound fluid. Wound fluid was preincubated with inhibitor for 30 min at 4 C before the addition of [125I]insulin and incubation for 90 min at 37 C. Degradation was measured by precipitation in 10% TCA. ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Approximately 15% of all patients with diabetes will develop a peripheral ulcer (17, 18). Twenty percent of all patients with diabetes admitted to a hospital will have a skin ulcer. The risk of amputation in a patient with diabetes is 15–40 times higher than that in a patient without diabetes. The presence of an ulcer in a diabetic patient has a profound impact on the quality of life for the patient and on the delivery of care. The cost of care for diabetic ulcers and the associated amputations is staggering (19). Although the prevalence of chronic ulcers has been estimated to be 120/100,000 people between 45–64 yr of age, the prevalence increases to more than 800/100,000 people over the age of 75 yr (20). Persons with diabetes have up to a 40-fold greater risk of lower extremity amputation than their nondiabetic counterparts (21). There were approximately 86,000 hospital discharges for diabetes-related nontraumatic amputations in the United States in 1996, the last year for which we have published statistics. The 5-yr survival rate after amputation of a diabetic limb is less than 50%. These grim statistics reflect an increased prevalence of peripheral lesions in diabetes, but also delayed healing.

The healing of peripheral wounds in patients with diabetes is a very complex process (2, 3, 4). Inadequacy of blood supply, infection, and repeated trauma are the three most important obstacles to successful healing. Wound healing also requires an adequate supply of growth-promoting materials. Numerous growth factors are important in wound healing (22). Platelet-derived growth factor (PDGF), fibroblast growth factor, and epidermal growth factor (23) have been shown to accelerate tissue repair in an experimental wound model (24). These agents applied locally resulted in a 2-fold increase in reepithelialization of the wound. Other combinations of growth factors had significant effects on wound healing (22, 24). In human studies, recombinant PDGF had significant beneficial effect on wound healing (25). This approach is now accepted therapy for the treatment of peripheral ulcers. Insulin is one of the primary anabolic hormones in the body, and numerous studies have shown beneficial effects of insulin therapy on wound healing (26, 27). Insulin increases wound tensile strength (27) and stimulates protein anabolism in skin and muscle (28).

Studies in animals with experimental wounds have defined some abnormalities due to insulin and growth factor action and metabolism. Insulin resistance is a consistent finding, which may be due to postreceptor abnormalities (29, 30) or increased insulin degradation in wound fluid. Growth factor degradation is also increased in experimental wound fluid (31).

Timely healing moves through a precise stepwise progression dependent on communication and precise interaction between multiple cell types (e.g. platelets, neutrophils, macrophages, fibroblasts, endothelial cells, and epithelial cells). The communication is carried out by chemical mediators, such as growth factors (e.g. PDGF, insulin, epidermal growth factor, TGF, and keratinocyte growth factor) (32) and cytokines (e.g. ILs and TNF). Analysis of wound fluid from both healing wounds and nonhealing wounds shows striking differences. Nonhealing wounds contain higher levels of inflammatory cytokines, fewer active growth factors, and higher levels of proteases. It can be postulated that inadequate growth factors may be partially attributable to the presence of the excessive proteases. Matrix metalloproteases have been shown to be harmful to the wound-healing process, with concentrations of matrix metalloproteases increased up to 65-fold in biopsies of diabetic foot ulcers (33, 34). Proteases are necessary in appropriate quantities and at the appropriate times for proper wound healing. Excessive proteolytic activity may be harmful.

Insulin-degrading activity is present in experimental wounds in animals (7), but no previous study has examined this activity in human wounds. In the present study we show that insulin-degrading activity is present in human wound fluid, and that the activity is higher in subjects with diabetes. The biochemical properties of the wound fluid insulin-degrading activity were consistent with the properties of the insulin-degrading enzyme (insulysin). Future work would include analysis of insulin degradation products by HPLC and immunoprecipitation to verify the action of insulin-degrading enzyme (14, 15) as well as studies of insulin and other growth factors in the wound fluid and the circulation.

Although it seems highly likely that the insulin-degrading activity is due to IDE, the increased degrading activity could be a result of a lack of inhibitors of IDE, as has been described previously (35). Future studies in this area should include both specific identification of IDE and quantitative analysis by immunological or other apparatus. Insulin-degrading activity correlates with HbA1c levels, suggesting a mechanism for the relationship between glucose control and wound healing. Of considerable interest is that there was a strong correlation among insulin-degrading activity, wound healing, and HbA1c levels. Improvement in glucose control is a critical factor in wound healing, but a reduction in wound fluid insulin-degrading activity is also a potential therapeutic approach.


   Footnotes
 
This work was supported in part by a Veterans Affairs Merit Review (to W.C.D.).

Abbreviations: HbA1c, Hemoglobin A1c; IDE, insulin-degrading enzyme; NEM, N-ethylmaleimide; PDGF, platelet-derived growth factor; TCA, trichloroacetic acid; V.A.C., Vacuum-Assisted Closure.

Received August 5, 2003.

Accepted November 10, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. National Diabetes Data Group 1995 Diabetes in America, vol 2. Bethesda, MD: NIH; Publication 95-1468
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