Molecular and Cellular Biochemistry 270: 71–77, 2005. c�Springer 2005

Energy metabolism in the granulation tissueof diabetic rats during cutaneous wound healing

Asheesh Gupta and Ram RaghubirDivision of Pharmacology, Central Drug Research Institute, Lucknow, India

Received 27 February 2004; accepted 28 September 2004

Abstract

The skin cells chiefly depend on carbohydrate metabolism for their energy requirement during cutaneous wound healing.Since the glucose metabolism is greatly hampered in diabetes and this might affect wound repair process. This prompted usto investigate the intermediate steps of energy metabolism by measuring enzyme activities in the wound tissues of normaland streptozotocin-induced diabetic rats following excision-type of cutaneous injury. The activities of key regulatory enzymesnamely hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6phosphate dehydrogenase (G6PD) have been monitored in the granulation tissues of normal and diabetic rats at different timepoints (2, 7, 14 and 21 days) of postwounding. Interestingly, a significant alteration in all these enzyme activities was observedin diabetic rats. The activity of PFK was increased but HK, LDH and CS showed a decreased activity in the wound tissue ofdiabetics as compared to normal rats. However G6PD exhibited an elevated activity only at early stage of healing in diabeticrats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the wound tissueof diabetic rats may affect the energy availability for cellular activity needed for repair process and this may perhaps be one ofthe factor responsible for impaired healing in these subjects. (Mol Cell Biochem 270: 71–77, 2005)

Key words: energy metabolism, cutaneous, wound, diabetes, impaired healing

Introduction

The process of wound healing requires increased mitotic andsynthetic activities on the part of the cells adjacent to the siteof injury. The skin cells undergo rapid cell division duringprocess of wound repair and this is accompanied by a greaterdemand of metabolic energy. The skin presents an active sitefor carbohydrate metabolism [1, 2]. It has been demonstratedthat the proliferating epithelial cells as well as wounded skinare primarily dependent on the glycolytic pathway for energyrequirement [2–5]. The contribution of Krebs cycle to glu-cose metabolism seems relatively less in wounded tissue thanin normal skin. However, a high activity of pentose phosphatepathway has been shown in repairing epithelium suggesting

Address for offprints: R. Raghubir, Division of Pharmacology, Central Drug Research Institute, P.O. Box No. 173, Lucknow 226001, India(E-mail: raghubirr@yahoo.com)Present address: A. Gupta, Division of Biochemical Pharmacology, Defence Institute of Physiology and Allied Sciences, Delhi 110054, India.

its crucial role in the synthesis of the carbon backbone essen-tial for nucleic acid synthesis [2].

Wound healing is a dynamic and interactive process, com-prising three overlapping phases i.e. inflammation, gran-ulation tissue formation and remodeling. Though healingprocess takes place by itself but most healing failures areassociated with some form of impairment including infec-tion, diabetes, immunosuppression, obesity and malnutrition[6]. Diabetes causes impaired wound healing owing to sev-eral factors, primarily related to defects in the inflamma-tory response because of impaired granulocytic function andchemotaxis [7]. Other abnormalities associated with woundhealing of diabetic individuals include impaired neovascular-ization, a decreased synthesis of collagen, increased levels of

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proteinases and defective macrophage function [8, 9]. How-ever, the exact mechanisms for impaired healing of woundsin diabetics is still poorly understood.

The glucose metabolism is greatly hampered in diabeticmellitus. The maximal enzyme activities associated with en-ergy metabolism have been observed during wound healing ofnormal subjects. In addition, it has been shown that enhancedactivities of glycolytic enzymes in regenerated tissues seem tocontribute an increased cellular demand for energy require-ment during healing of acute wounds [2, 5, 10]. However,their status in the wound tissue of diabetic subjects have notbeen so far investigated. This prompted us to study the profileof key enzyme activities associated with energy metabolismduring cutaneous wound healing in diabetic rats.

Materials and methods

Chemicals and enzymes

Streptozotocin (STZ), phosphoenolpyruvate (PEP), pyruvatekinase and dithionitrobenzoic acid (DTNB) were purchasedfrom Sigma (St. Louis, MO, U.S.A.). All other chemicals andenzymes used in the experiments were obtained from SRL(Bombay, India).

Animals

Adult male Sprague-Dawley rats, weighing 200–250 g wereobtained from the National Laboratory Animal Centre of theInstitute. The animals were fed a standard rat chow diet andhad ad libitum access to water. The experiments were per-formed in accordance with regulations specified by the insti-tutional ethics committee.

Induction of diabetic state

The study was conducted in normal and streptozotocin (STZ)induced diabetic rats. Basal blood glucose level of rats wasmeasured using a glucometer (Ames, Bayer Diagnostic, In-dia). Next day after overnight fasting, the animals were in-jected with single dose of streptozotocin (50 mg/kg, i.p.)in 0.1 M citrate buffer, pH 4.5 to produce diabetes. Con-trol rats received buffer alone by the same route. The fol-lowing day, the blood glucose level of all animals in thediabetic group was monitored and animals showing bloodglucose level nearly three-folds higher as compared to con-trol value were considered diabetic. The blood glucose levelremains maintained throughout the course of experiment.The animals were kept for 15 days to stabilize the diabeticcondition.

Wound creation and excision

Animals were anesthetized with ether and hair on the dorsalsurface were shaved off and underlying skin cleaned with70% ethanol. Four full thickness wounds (8-mm in diame-ter) were made on the back of these rats by excising skinand the underlying panniculus carnosus using skin biopsypunch (Acuderm Inc. Lauderdale, FL, U.S.A.), under asep-tic condition. Two punch wounds on either side of dorsalline in each rat, were created as described earlier [6]. Ani-mals were allowed to recover and housed individually in thesterile metallic cages, maintained at standard animal houseconditions. Wound tissue excision from normal and diabeticrats was done after sacrificing by an overdose of anestheticether on 2nd-, 7th-, 14th- and 21st-day postwounding usingthe biopsy punch [6]. Sixteen rats have been taken at eachtime points of study. The skin punches were also collectedfrom non-wounded animals of both groups.

Preparation of tissue homogenate

Samples for the estimation of enzyme activities associatedwith energy metabolism were prepared as described earlier[11]. All operations were performed at 4 ◦C unless mentionedspecifically. Briefly, a 20% homogenate was made in chilled0.15 M KCl containing 5 mM EDTA using Polytron Homoge-nizer (PT 3100, Switzerland). Each sample was homogenizedby giving four strokes of 15 s. duration. Samples were son-icated (10 bursts of 5 s each at 5 s gap between two bursts)by using Ultra-Sonicator (W-385, Heat System UltrasonicsInc., U.S.A.). The supernatant was collected for estimationof enzyme activities following centrifugation at 9000 × g ina refrigerated centrifuge for 20 min.

The assays of all the enzyme activities were performedat 25 ◦C. The methodology was designed to ensure maximalenzyme activities in the crude samples. The results of en-zyme activities were expressed as n mol/min/g fresh weightof granulation tissue.

Assay of enzyme activity

HexokinaseHexokinase (HK, E.C. 2.7.1.1) activity was measured by thecoupled enzyme assay system as described by Supowit andHarris [12], using glucose as a substrate. The sample wasmixed with assay buffer consisting of 50 mM triethanolamineand 10 mM MgCl2 (pH 7.4), 1M glucose, 50 mM NADPand 140 U/mL of glucose 6-phosphate dehydrogenase. Thereaction was initiated by the addition of 200 mM ATP, and therate of NADP reduction was recorded at 340 nm for 3 min.Calculation of enzyme activity was based on the reduction ofNADP (∈NADP = 6220 M−1 cm−1).

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PhosphofructokinasePhosphofructokinase (PFK, E.C. 2.7.1.11) activity was mea-sured by the method of Bergmeyer et al. [13], using fructose-6phosphate as a substrate. The sample was added to 50 mMTris-HCl buffer (pH 8.0), 500 mM MgCl2, 1 M KCl, 100mM ATP, 100 mM fructose-6 phosphate, 10 mM NADH, 50mM phosphoenolpyruvate and 75 U/mL of pyruvate kinaseplus 40 U/mL of lactate dehydrogenase in a quartz cuvette.The NADH metabolized was assessed at 340 nm by spec-trophotometry. PFK activity was calculated based on 1 µmolfructose 1,6 diphosphate = 2 µmol NADH consumed.

Lactate dehydrogenaseThe activity of Lactate dehydrogenase (LDH, E.C. 1.1.1.27)was determined according to the procedure of Korenber[14], in the presence of sodium pyruvate as a substrate. Thesample was mixed with LDH reaction solution containing10 mM sodium pyruvate in 100 mM sodium phosphate buffer(pH 7.0). Reaction was initiated by the addition of 4.22mM NADH and assayed at 340 nm for 3 min. The calcu-lation of enzyme activity was based on oxidation of NADH(∈NADH= 6220 M−1 cm−1).

Citrate synthaseCitrate synthase (CS, E.C. 4.1.3.7) activity was measuredusing the method of Shepherd and Garland [15]. The methodwas based on chemical coupling of Coenzyme A (CoA-SH),released from acetyl-CoA to DTNB. The sample was added to100 mM Tris-HCl buffer (pH 8.0), 10 mM DTNB and 5 mMacetyl-CoA. Reaction was initiated by the addition of 50 mMoxaloacetate. The rate of change in extinction was measuredat 412 nm. Blank contained no acetyl-CoA. Calculation ofenzyme activity was based on ∈ = 5.4 cm2/µ mole.

Glucose-6 phosphate dehydrogenaseGlucose-6 phosphate dehydrogenase (G6PD, E.C. 1.1.1.49)assay was done following the method of Lohr and Waller [16],using glucose-6 phosphate as a substrate. The sample wasmixed with assay buffer consisting of 50 mM triethanolaminebuffer plus 5 mM EDTA (pH 7.5) and 30 mM NADP. Re-action was initiated by the addition of 42.6 mM glucose-6phosphate. The G6PD-dependent reduction of NADP wasmeasured as the increase in A340. The calculation of enzymeactivity was based on reduction of NADP (∈NADH = 6220M−1 cm−1).

Statistical analysis

The results were expressed as the mean ± S.E.M. Statisticalanalysis was performed using Student’s t-test to compare theenzyme activities between normal and diabetic wound tis-sue at respective time points. ANOVA followed by post hoc

(Dunnett’s test) was used to compare the enzyme activities ofthe skin (day 0) with regenerated wound tissue at various timepoints. The value of p < 0.05 was considered as significant.

Results

Enzyme activities associated with energy metabolismduring cutaneous wound healing in normal rats

Three glycolytic enzymes (HK, PFK, LDH) and one Krebscycle enzyme (CS) assayed in regenerated wound tissues ofnormal rats exhibited, a significant increase in the activitiesat different time points of healing as compared to their statusin the skin. However, G6PD of pentose phosphate pathwaydemonstrated a decreased activity during the period of obser-vation.

There was a maximal increase in the PFK activity by 370%on day 2 postwounding (Fig. 2). The HK, LDH as well as CSactivities were found significantly elevated on day 7 post-wounding (Figs. 1, 3 and 4). The HK and LDH activitiesreturned to basal level but PFK and CS activities remainedelevated even on day 21 postwounding. The G6PD activitywas significantly reduced on day 2 postwounding and whichgradually tended to partly recover at later days of healing(Fig. 5).

Enzyme activities associated with energy metabolismduring cutaneous wound healing in diabetic rats

The trend of glycolytic enzyme activities was very similarin wound tissue of diabetic rats as seen in normal rats. Themaximal HK activity was observed on day 7 which returnedto basal level (diabetic skin) on day 21 postwounding (Fig. 1).

The PFK exhibited maximal increase on day 2 and whichremained elevated by about 2 fold on day 7 and 21 postwound-ing (Fig. 2). Further, the LDH activity was found significantlyelevated during the period of observation (Fig. 3). Citrate syn-thase activity was significantly increased on day 7 and whichremained elevated on later days of postwounding (Fig. 4).However, G6PD registered a significant decreased activityon 7 to 21 days postwounding in the regenerated tissues ofdiabetic rats (Fig. 5).

Comparison of enzyme activities in the regenerated tissuesof normal and diabetic rats

All enzymes demonstrated significant alterations in their ac-tivities in the wound tissue of diabetic rats as comparedto the tissue obtained from normal rats on the respectivedays. The HK exhibited significant decreased activity in the

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Fig. 1. Hexokinase activity in the skin (0 day) and regenerated wound tissuesof normal and diabetic rats. Values are mean ± S.E.M. of 16 rats at eachtime point. Significance (∗p < 0.05, ∗∗p < 0.01) as compared to the skinwith regenerated wound tissue. Significant difference (bp < 0.01) betweennormal and diabetic wound tissue at respective time points.

Fig. 2. Phosphofructokinase activity in the skin (0 day) and regeneratedwound tissues of normal and diabetic rats. Values are mean ± S.E.M. of16 rats at each time point. Significance (∗∗p < 0.01) as compared to theskin with regenerated wound tissue. Significant difference (ap < 0.05, bp <

0.01) between normal and diabetic wound tissue at respective time points.

regenerated tissues at different time points of postwoundingin diabetic rats (Fig. 1). On the other hand PFK exhibited anelevated activity with maximal increase on day 2 postwound-ing, which remained elevated during later days of postwound-ing (Fig. 2). LDH and CS demonstrated a decreasing pattern

Fig. 3. Lactate dehydrogenase activity in the skin (0 day) and regeneratedwound tissues of normal and diabetic rats. Values are mean ± S.E.M. of16 rats at each time point. Significance (∗∗p < 0.01) as compared to theskin with regenerated wound tissue. Significant difference (ap < 0.05,bp < 0.01) between normal and diabetic wound tissue at respective timepoints.

Fig. 4. Citrate synthase activity in the skin (0 day) and regenerated woundtissues of normal and diabetic rats. Values are mean ± S.E.M. of 16 rats ateach time point. Significance (∗∗p < 0.01) as compared to the skin with re-generated wound tissue. Significant difference (bp < 0.01) between normaland diabetic wound tissue at respective time points.

in the wound tissues of diabetic as compared to the normalrats (Figs. 3 and 4). However, the G6PD exhibited an ele-vated activity on days 2 and 7 postwounding in diabetic ascompared to normal rats (Fig. 5).

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Fig. 5. Glucose 6-phosphate dehydrogenase activity in the skin (0 day) andregenerated wound tissues of normal and diabetic rats. Values are mean ±S.E.M. of 16 rats at each time point. Significance (∗∗p < 0.01) as comparedto the skin with regenerated wound tissue. Significant difference (bp < 0.01)between normal and diabetic wound tissue at respective time points.

Discussion

Cellular energy is the key requirement for biosynthetic activ-ity during healing process. Wounded skin exhibits increasedmetabolic activity essential to maintain the homeostatic dy-namic equilibrium in the altered environment. It has beenshown that the utilization of glucose through anaerobic gly-colysis is the main energy pathway in the skin [10]. An in-crease in glucose utilization has been observed in repairingepithelium during healing of skin [2], in the psoriatic skin[17] and in growing hair follicles [18].

The results of present study clearly indicate that healingprocess significantly enhanced the HK, PFK, LDH and CSenzyme activities associated with energy metabolism in gran-ulation tissues of normal rats. Both HK and PFK act as arate limiting enzymes of glycolysis. The most significantchange was an enhancement of glycolytic enzyme activitiesin general, being maximal increase in PFK activity. At theearly stage of healing, there is a marked gradient of oxygenwithin wound, and central portion of the wound is relativelyhypoxic [19, 20]. Therefore, elevated activity of LDH, anenzyme of anaerobic glycolysis is indicative of various cel-lular components of granulation tissue at the early stagesof healing, which is metabolically adopted for survival in ahypoxic environment. Furthermore, LDH activity regain itsbasal level within 3 weeks after cutaneous injury suggesting

that new capillary network has been established by this timeand wound environment has been changed from hypoxic tonormoxic state. Moreover, it has also been found that lactate,the end product of glycolysis, influence the proline hydrox-ylase activity, which enhances the rate of collagen formation[21]. Similar findings of increased glucose utilization andlactate formation have already been observed in human skingrafts [22].

The maximal activity of CS suggests that a transient in-crease in the potential for aerobic metabolism has occurredin the healing wound following excision-type of cutaneousinjury. Moreover, the increase in the CS activity could also re-flect an increase in the capacity for lipogenesis from glucoseto prevent water loss from the healing skin [23]. It has beenshown that the maximal activities of HK and PFK provide aquantitative index of the glycolytic flux [24, 25]. While themaximal activities of LDH and CS provide a qualitative in-dex of the flux through the breakdown and synthesis of lactateand Krebs cycle [10].

Interestingly, alterations in the glycolytic enzyme activi-ties were confined only in the granulation tissue as revealedby skin punch taken from non-wounded site (personal obser-vation). Thus, it appears that an increase in the activities ofglycolytic enzymes might be providing a metabolic flux justwithin wound environment.

In the present study a depleted activity of G6PD has beenobserved following 21 days of postwounding suggesting thatNADPH, an important reducing agent which is generated byG6PD activity, seems to be adversely affected during cuta-neous injury. The NADPH maintains cellular redox potentialduring oxidative stress. Further, a depleted level of antioxi-dants has been observed during healing of a cutaneous wound,which recovers as the healing progresses [26]. Therefore, itappears that depleted antioxidant levels might be due to de-creased activity of G6PD. Moreover, a minimal alteration inG6PD activity in granulation tissue has also been reportedearlier, which indicates that NADPH is important in epider-mal regeneration and keratinization [27, 28].

Our results also indicate a significant increase in the ac-tivities of glycolytic enzymes in the granulation tissue ofdiabetic rats, suggesting that the capacity to utilize glucoseincreased in these subjects during wound healing as observedin normal rats. However, the profile of all these enzyme ac-tivities were significantly altered in diabetic as compared tonormal animals at respective time points of postwounding. Ithas been shown that an impaired HK activity in diabetic ratsmight be due to defective glucose transport or phosphoryla-tion [29–31]. The other possibility of decreased activity ofHK may be due to loss in the amount of HK protein ratherthan to an inhibition by glucose-6 phosphate (G-6P) or othermetabolites [31]. Furthermore, it has been already shown thatin diabetic animals the HK activity in muscle, liver, heart, andother insulin sensitive tissue is also markedly impaired [32].

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However, insulin administration in diabetic rats increases theHK activity, and that might be due to interconversion of in-active enzyme into active form [31, 33].

In the present study, an increased activity profile of PFKand decreased activity of CS was observed at different timepoints of postwounding in diabetic rats. Interestingly, Liuet al. [34, 35] have also reported similar type of results us-ing rat islets cultured with high glucose and long fatty acids,a condition simulating to a diabetic state. They further sug-gested that G-6P, produced by HK is in equilibrium withfructose-6 phosphate (F-6P), and therefore the level of G-6Pis governed by PFK. Elevated PFK activity is due to in-creased level of fatty acids, which lowers the cellular levelof G-6P and deinhibits HK activity. Citrate is a potent in-hibitor of PFK and reduction in citrate would act in tandemwith the increased PFK activity and thus also affects G-6Pcatabolism.

An increased G6PD activity has been found at early stageof healing in diabetic rats, suggesting that redox potentialmight be altered due to increased oxidative stress during thistime period and thereby induced the activity of G6PD, whichproduced NADPH as a reducing agent. Interestingly, similarchanges in the G6PD activity were also observed in immuno-suppressed wounds [11].

It has been shown that lymphocytes and macrophages, themain immune cellular components in the healing process,having high capacity to utilize glucose as main energy source[5, 25]. Further, it has been shown that diabetes causes defectin the inflammatory responses [7]. Therefore, circulation ofimmune cells such as neutrophils, macrophages and fibrob-lasts to the wound bed is impaired, which may affect theenergy availability for biosynthetic processes necessary forrepair. Thus, the observed differences in the enzyme activ-ities between normal and diabetic rats could be explainedby impaired infiltration of immunocytes at the wound site indiabetic rats but this needs further investigation.

Thus, the results of present investigation indicate that al-tered enzyme activities associated with energy metabolismmay perhaps be one of the factor responsible for impairedwound healing in diabetic rats.

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