Jeffry D. Shearer

Insulin is degraded extracellularly in wounds by insulin-degrading enzyme (EC 3.4.24.56)

The exact mechanism by which insulin reverses impaired wound healing is unknown. Previous investigators have shown that insulin is degraded in experimental wounds, suggesting that the action of insulin may be locally modified. The following study corroborates these findings and identifies the major proteinase responsible for insulin degradation in wound fluid (WF). Adult male Fisher rats were wounded by subcutaneous implantation of polyvinyl alcohol sponges while under pentobarbital sodium anesthesia. WF and serum were collected on 1, 5, 10, and 14 days postinjury. Decreased insulin concentration in late WF correlated with an increased insulin-degrading activity. Multiple proteinases appear to participate in the overall degradation of insulin in WF. However, the primary enzyme responsible for insulin degradation in WF was characterized by immunoprecipitation and immunoblotting and identified as the neutral thiol-dependent metalloproteinase, insulin-degrading enzyme (EC 3.4.24.56). Exogenous steroid administration caused a decrease in WF insulin-degrading activity. Glucagon and adrenocorticotrophin degradation was also observed, whereas minimal degradation of insulin-like growth factors I and II and epidermal growth factor was detected in WF. The ability to extracellularly degrade insulin may represent a unique mechanism for the regulation of this hormones role in healing wounds.The exact mechanism by which insulin reverses impaired wound healing is unknown. Previous investigators have shown that insulin is degraded in experimental wounds, suggesting that the action of insulin may be locally modified. The following study corroborates these findings and identifies the major proteinase responsible for insulin degradation in wound fluid (WF). Adult male Fisher rats were wounded by subcutaneous implantation of polyvinyl alcohol sponges while under pentobarbital sodium anesthesia. WF and serum were collected on 1, 5, 10, and 14 days postinjury. Decreased insulin concentration in late WF correlated with an increased insulin-degrading activity. Multiple proteinases appear to participate in the overall degradation of insulin in WF. However, the primary enzyme responsible for insulin degradation in WF was characterized by immunoprecipitation and immunoblotting and identified as the neutral thiol-dependent metalloproteinase, insulin-degrading enzyme (EC 3.4.24.56 ). Exogenous steroid administration caused a decrease in WF insulin-degrading activity. Glucagon and adrenocorticotrophin degradation was also observed, whereas minimal degradation of insulin-like growth factors I and II and epidermal growth factor was detected in WF. The ability to extracellularly degrade insulin may represent a unique mechanism for the regulation of this hormones role in healing wounds.

Continuity between wound macrophage and fibroblast phenotype: analysis of wound fibroblast phagocytosis

Analysis of phagocytic activity in wound fibroblasts was chosen as a means to assess the possible continuity between macrophage and fibroblast phenotypes. Fibroblast phagocytosis of uncoated, IgG-coated, or collagen-coated fluorescent beads was analyzed by flow cytometry in vivo and in vitro. Phagocytosis of fluorescent beads by procollagen I-positive cells (fibroblasts) was evaluated in vivo by injecting beads into subcutaneously implanted sponge wounds in anesthetized Fisher rats. Phagocytic activity of a purified population of wound fibroblasts was measured in vitro and correlated with oxidation state using hydroethidium. In the wound environment, 50-60% of the cells that engulfed uncoated, IgG-coated, or collagen-coated beads were procollagen I-positive cells (i.e., fibroblasts). Procollagen I-positive cells engulfed uncoated and IgG-coated beads in preference to collagen-coated beads in vivo. Cultured wound fibroblasts engulfed uncoated, IgG-coated, and collagen-coated particles. The majority of fibroblasts that engulfed beads were in an elevated oxidation state. We conclude that substantial fibroblast phagocytosis occurs in the wound, but scavenger receptor-mediated fibroblast phagocytosis is different from that of macrophages. Additional markers will be helpful in defining the macrophage fibroblast continuum.Analysis of phagocytic activity in wound fibroblasts was chosen as a means to assess the possible continuity between macrophage and fibroblast phenotypes. Fibroblast phagocytosis of uncoated, IgG-coated, or collagen-coated fluorescent beads was analyzed by flow cytometry in vivo and in vitro. Phagocytosis of fluorescent beads by procollagen I-positive cells (fibroblasts) was evaluated in vivo by injecting beads into subcutaneously implanted sponge wounds in anesthetized Fisher rats. Phagocytic activity of a purified population of wound fibroblasts was measured in vitro and correlated with oxidation state using hydroethidium. In the wound environment, 50-60% of the cells that engulfed uncoated, IgG-coated, or collagen-coated beads were procollagen I-positive cells (i.e., fibroblasts). Procollagen I-positive cells engulfed uncoated and IgG-coated beads in preference to collagen-coated beads in vivo. Cultured wound fibroblasts engulfed uncoated, IgG-coated, and collagen-coated particles. The majority of fibroblasts that engulfed beads were in an elevated oxidation state. We conclude that substantial fibroblast phagocytosis occurs in the wound, but scavenger receptor-mediated fibroblast phagocytosis is different from that of macrophages. Additional markers will be helpful in defining the macrophage fibroblast continuum.

Macrophage interaction with skeletal muscle: a potential role of macrophages in determining the energy state of healing wounds

A decrease in ATP and creatine phosphate (CP) is characteristic of local injury to skeletal muscle. Recent data have suggested adequate potential for high-energy phosphate production in the wounded tissue. Thus, an adequate explanation for the deficit in the high-energy tissue content in wounds was lacking. Since the wound has multiple components (muscle + cellular infiltrate), the tissue content represents the summation of these components. Therefore, a technique to separate these components was designed. Using a 0.5% solution of lambda-carrageenan as the wounding agent, the extensor digitorum longus muscles (EDL) of male Fisher rats were unilaterally wounded with intramuscular injections. Five days later, both wounded and contralateral nonwounded muscles were incubated in a standardized fashion. The groups of EDL were: wounded as described, contralateral nonwounded, or contralateral nonwounded with the addition to the incubate of 6 X 10(6)/ml lambda-carrageenan elicited peritoneal macrophages. Following incubation, the individual component parts of the system (muscle and macrophage) were rapidly frozen and assayed for high-energy phosphate and DNA content. Examination of the high-energy phosphate content of the separate components of a wound demonstrated that macrophages increased the ATP and CP content of normal skeletal muscle. Yet when total high-energy phosphate content was normalized for total DNA (muscle + macrophages) in the reconstituted system, the values approximated those of wounded muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Portal donor-specific blood transfusion and mycophenolate mofetil allow steroid avoidance and tacrolimus dose reduction with sustained levels of chimerism in a pig model of intestinal transplantation.

Background. In a pig model of intestinal transplantation, we previously showed that hepatic conditioning through portal donor-specific blood transfusion (pDSBT), high-dose tacrolimus (TAC), and steroids prevented rejection and increased survival. Our current study tests a protocol of pDSBT, short-term mycophenolate mofetil (MMF), and low-dose TAC to eliminate the use of steroids, reduce TAC dosage, and increase the level of chimerism in the peripheral blood. Materials and Methods. Four groups of outbred, mixed lymphocyte culture (MLC)-reactive pigs underwent bowel transplants and pDSBT. Immunosuppression (group 1, high-dose TAC and steroids; group 2, low-dose TAC and MMF; group 3, low-dose TAC, MMF, and aminoguanidine; group 4, low-dose TAC, MMF, and arginine) was discontinued after 28 days. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements. Chimerism levels were determined using a Q-PCR analysis. Results. Pig survival and death rates due to rejection did not significantly differ between the four groups. Chimerism levels determined by Q-PCR analysis were not different until day 28. After discontinuation of immunosuppression, we noted a trend (P =0.15) toward higher mean chimerism levels on day 60 for groups 2, 3, and 4 (9%) vs. group 1 (0.5%). Tissue cytokine and serum nitrate levels did not significantly differ between the four groups. Attempts to modify nitric oxide synthase activity offered no added benefit. Conclusions. The combination of pDSBT, MMF, and low-dose TAC (vs. high-dose TAC and steroids) allowed sustained levels of mixed chimerism to develop after discontinuation of immunosuppression.

Tumor Effects on Gluconeogenesis in the Isolated Perfused Rat Liver

Alterations in metabolism in the tumor-bearing host can be explained by: 1) alterations of metabolic processes in the tumor itself, and/or 2) tumor effects on host metabolism. Tumor effects on host liver metabolism were studied using an isolated perfused rat liver preparation. The livers of fasted female Lewis Wistar rats with and without transplanted subcutaneous mammary tumors were perfused for 1 hr with medium containing 5 mM glucose and physiological levels of amino acids. The rate of gluconeogenesis, as measured by conversion of 14C-lactate to 14C-glucose, showed a significant increase in the rate of glucose production from lactate in tumor-bearing rats (2.40 vs 2.00 mumol/min/100 gm). Hepatic glycogen and 14C-glycogen content were not significantly different between the two groups. In order to evaluate whether this tumor model exhibits characteristic changes in metabolism previously reported in other animal tumor models, serum lactate, triglyceride, glucose, and blood urea nitrogen were measured in non-perfused animals. The serum concentration of lactate and triglycerides were significantly higher in tumor-bearing rats (0.9 mM vs 2.7 mM lactate; 244 mg % vs 365.5 mg % triglycerides). Serum glucose and blood urea nitrogen were not significantly different in the two groups. An effect of tumor on host energy metabolism and serum metabolite levels is demonstrated. A method for the study of host-tumor metabolic interactions is described.

Mactinin treatment promotes wound-healing-associated inflammation in urokinase knockout mice

Mactinin, a 31 kDa fragment from the amino‐terminal end of α‐actinin, is chemotactic for monocytes and can promote monocyte/macrophage maturation. Macrophages are essential for wound healing, in which they play key roles in debridement, angiogenesis, fibroblast proliferation, and collagen metabolism. We have previously determined that urokinase is necessary to form mactinin from extracellular α‐actinin, which may be present at sites of inflammation as a result of cell movement. Thus, urokinase knockout mice are unable to form mactinin and therefore are an ideal model to study mactinins effects on wound healing. Saline‐ and mactinin‐treated wounds were analyzed in a subcutaneous sponge wound model in both wild‐type and urokinase knockout mice. The wounded urokinase knockout mice had markedly decreased leukocyte infiltration compared with wounded wild‐type mice. In addition, production of the proinflammatory cytokine, interleukin‐12, and of collagen was also decreased in knockouts. Treatment of knockout mice with mactinin resulted in leukocyte infiltration numbers, interleukin‐12 levels, and hydroxyproline measurements similar to those in wild‐type mice. The results suggest that impaired wound healing in urokinase‐deficient mice can be restored by administration of mactinin.

The temporal characteristics of the metabolic and endocrine response to injury.

The neuroendocrine and substrate responses immediately after injury have been extensively investigated in man and animals. The purpose of the present study was to examine simultaneously, the temporal, metabolic and endocrine consequences of a single uniform injury induced by the injection of lambda-carrageenan into the hindlimbs of male Sprague-Dawley rats and to compare this response to that observed in semistarved pair-fed control animals. Immediately after injury there was a decrease in the plasma hematocrit, increase in tissue water and peripheral vasoconstriction that suggested hypovolemia. This was followed by a restoration of the blood volume by 1 day as reflected in hemodilution. Alterations in insulin, glucagon, ACTH, corticosterone, epinephrine, norepinephrine, and dopamine in wounded animals occurred during the first 5 days. However, similar changes were observed in pair-fed control animals from days 1 to 5. These findings implied that the early endocrine response observed from 0 to 24 hours after injury arises, primarily as a result of hypovolemia, whereas the response observed from 1 to 5 days appeared to be the result of semistarvation. In contrast to the endocrine alterations observed, alterations in the plasma concentrations of lactate, acetoacetate and beta-hydroxybutyrate persisted for up to 15 days. The presence of these substrate alterations in the absence of hormonal stimuli suggest that nonendocrine mechanisms exist to induce these alterations. The possibility is raised that these substrate alterations may be, at least in part, the result of the inflammatory infiltrate.

Effect of starvation on the local and systemic metabolic effects of the λ-carrageenan wound☆☆☆

Abstract The contribution that starvation makes to the altered glucose metabolism in injured rats was evaluated. Food intake, weight change, nitrogen balance, and muscle tissue concentrations of glycogen, glucose, and the glycolytic intermediates were determined in these animals. This study concluded that the wounded and pair fed control groups presented adequately represent the metabolic states associated with injury and semistarvation in experimental animals, decreased food intake plays a major role in the weight loss and nitrogen balance in this wound model, wounding overrides two of the controlling steps of glycolysis (hexokinase and phosphofructokinase) in skeletal muscle during starvation, the finding of similar pyruvate dehydrogenase activity after wounding and starvation as demonstrated by tissue lactate to pyruvate ratios and lactate and pyruvate concentrations suggest that lactate production in wounded tissue may not be simply a manifestation of an altered redox state secondary to anaerobic conditions.

Somatomedin-A inhibits glucagon-stimulated hepatic gluconeogenesis.

Somatomedin/insulin-like growth factors have been noted to produce insulin-like actions on the liver that include the ability to inhibit the formation of glucagon-stimulated but not basal c-AMP production. This raises the possibility that these compounds may also be able to inhibit glucagon-stimulated hepatic gluconeogenesis. The purpose of this study was to determine the effects of varying concentrations of somatomedin-A on glucagon-stimulated gluconeogenesis in isolated perfused rat liver. The infusion of glucagon increased the rate of gluconeogenesis from 0.0038 +/- 0.001 to 0.042 +/- 0.007 microM 14C-glucose made from 14C-alanine per min. This action of glucagon was reduced to 22% of its maximum by the coinfusion of 1 microU/ml of SM-A and completely eliminated by the coinfusion of 100 microU/ml of SM-A. Somatomedin alone did not alter the basal rate of gluconeogenesis. The decrease in the release of 14C glucose from livers that had been stimulated by glucagon could not be attributed to increased glycogenolysis. Thus, it appears that the reduction in 14C release represents SM-A mediated reduction in glucagon-stimulated gluconeogenesis.