ORIGINAL ARTICLE

Expression of leptin and its long-form receptor in the marginalcutaneous tissues of diabetic foot ulcers

Ying Cao • Fang Gao • Chen-Zhong Li •

Yao-ming Xue

Received: 3 April 2012 / Accepted: 27 August 2012

� Springer-Verlag 2012

Abstract To investigate the relationship between the

expression of leptin and its long-form receptor, OB-RL,

and wound healing in diabetic foot ulcers. Biopsies from 10

patients with diabetic foot ulcers (DU group), 10 with non-

diabetic foot ulcers (NDU group), and 10 with normal skin

(normal control, NC group) were examined. Leptin and

OB-RL mRNA and protein levels were assessed using RT-

PCR and immunohistochemistry analyses, respectively.

The cuticle thickness was significantly greater, and the

epidermal layer was significantly lesser in the DU and

NDU groups. Leptin protein expression was significantly

higher in the DU and NDU than NC group (P \ 0.001),

whereas OB-RL mRNA and protein expressions were

significantly lower in the DU group and significantly higher

in the NDU group (P \ 0.001). Diabetic foot ulcer duration

was negatively correlated with OB-RL protein expression

(q = -0.671, P = 0.034). Decreased OB-RL may result in

reduced leptin signaling in diabetic foot ulcers. Further

studies are required to determine whether OB-RL levels are

related to the prognosis of diabetic foot ulcers, as well as to

explore the use of leptin or mimetics for promoting ulcer

healing.

Keywords Leptin � Diabetes � Ulcer

Introduction

Diabetic foot ulcers (DFUs) are a common complication of

diabetes mellitus, often resulting in limb amputation; the

incidence reaches 25 % in Western countries [1, 2]. With

the increasing incidence of diabetes mellitus worldwide,

determining the mechanism by which diabetic ulcers occur

and seeking new means of prevention and treatment is

imperative [3].

Although the way in which diabetes affects ulcerative

wound surface healing is not yet fully known, studies have

revealed that the ulcer-healing rate among non-diabetic

patients is greater, with 9.4-fold higher than that observed

for diabetic patients, despite receiving the same medication

[4]. Furthermore, in addition to cells within the ulcer,

differential cytokine and receptor expression, altered signal

transduction, and adipokines, including adiponectin, tumor

necrosis factor-a (TNFa), interleukin-6 (IL-6), and leptin,

may play an important role in the regulation of wound

healing [5, 6]. For example, leptin increases re-epithelial-

ization and accelerates wound healing [7]. Impaired wound

closure and contraction was observed in excisional wounds

treated with leptin neutralizing antibodies [5].

Because previous studies have proposed low leptin level

is responsible for poor wound healing [5, 7, 8], the present

study aimed to test the hypothesis that cutaneous leptin and

OB-RL expressions in patients with DFUs are lower than

that in patients with non-DFUs and normal patients,

thereby impeding wound healing. Studies analyzing the

expression of leptin and its receptor, OB-RL, in human

DFUs are rare, and none has been undertaken in Chinese

patients [9–17]. In this study, marginal cutaneous tissues

from DFUs, non-DFUs, and normal tissue were analyzed

for leptin and OB-RL mRNA and protein expression using

RT-PCR and immunohistochemistry, respectively.

Communicated by Renato Lauro.

Y. Cao � F. Gao � C.-Z. Li � Y. Xue (&)

Department of Endocrinology, Nanfang Medical University,

Guangzhou 510150, China

e-mail: yaomingxue@126.com

123

Acta Diabetol

DOI 10.1007/s00592-012-0428-8

Materials and methods

Study design

Samples were obtained from the marginal cutaneous tis-

sues of DFUs of 10 patients hospitalized from May to

December 2005 and in June 2006 in the Department of

Endocrinology, Nanfang Hospital Affiliated with the

Southern Medical University. The study was approved by

the Institutional Review Board of Nanfang Medical Uni-

versity and all patients provided written consent.

Biopsies were obtained from 10 patients with DFUs (DU

group), 10 with non-DFUs (NDU group), and 10 with

normal skin (normal control, NC group). Chronic wounds

were defined as those lasting longer than 1 month without

indication of healing despite conservative treatment. The

samples in the NDU and NC groups were matched in age

and gender with the DU group. In the DFU group, all

patients (3 females, 7 males) were diagnosed with type II

diabetes mellitus using the diagnosis standard formulated

by the WHO in 1999. In the NDU group, patients (6 males,

4 females) were hospitalized with non-diabetic chronic

dermal ulcers between December 2004 and March 2006.

Patients in the NDU group had no history of diabetes,

normal fasting blood glucose level, and the presence of a

chronic wound (Table 1). In the NC group, biopsies of

normal leg dermal tissues were obtained from patients

(7 males, 3 females), who underwent skin transplantation

during traumatic orthopedic surgery. Patients in the NC

group had no history of diabetes and normal fasting blood

glucose level. As shown in Table 1, no differences in age

and ulcer duration were observed.

The following exclusion criteria were used for the

present study: (1) the presence of malignant ulcerative skin

lesions and infected lesions (e.g., tuberculosis, fungal, and

tetanus lesions), (2) the presence of severe cardiac (NYHA

class III or above), hepatic (serum albumin \25 g/L), or

renal (creatinine C445 lmol/L and blood urea nitro-

gen C20 mmol/L) dysfunctions; (3) dexamethasone treat-

ment; (4) the presence of simple neurogenic ulcers; and (5)

ABI B0.7.

Immunohistochemical analysis

In addition to hematoxylin and eosin (H&E) staining,

immunohistochemical staining was performed using rabbit

anti-human OB-RL polyclonal antibodies (BA1234; Boster

Co.,Wuhan, Hubei, China), rabbit anti-human leptin poly-

clonal antibodies (sc-842; Santa Cruz Biotechnology, Santa

Cruz, CA), a streptavidin–biotin-peroxidase complex (SP)

staining kit and 3,3’-diaminobenzidine tetrahydrochloride

(DAB) (both from Beijing Zhongshan Biological Tech-

nology Co., Beijing, China) following manufacturer’s

instructions. The image analysis software, Image Tool 3.0

(UTHSCSA, TX, USA), was used to measure epidermal

and dermal thickness following microscopy.

Leptin- and OB-RL-positive cells were identified as

cells with the membrane and/or cytoplasm stained brown to

dark brown. Ten fields were randomly selected under an

optical microscope at 4009 magnification, and 100 cells in

each field were observed. The positive cell number was

counted, and the positive rate was calculated. The results

were divided into the following four categories based on

staining strength: negative (-; no staining detected in the

field), weakly positive (?; positive rate \10 %), positive

(??; positive rate 11–30 %), and strongly positive (???;

positive rate [30 %).

RT-PCR analysis

The following PCR primers were prepared for subsequent

use: leptin: sense, 50-ggctttggccctatcttttc-30 and antisense,

Table 1 Summary for subjects’ characteristics (n = 10 per group)

NC NDU DU P value

Agea (y) 56.5 (53.0, 66.0) 61.2 (58.1, 67.0) 59.1 (56.7, 60.9) 0.472

Genderb

Female 3 (30 %) 4 (40 %) 3 (30 %) 1.000

Male 7 (70 %) 6 (60 %) 7 (70 %)

Length of diabetes mellitusa (y) – – 9.7 (7.9, 11.2)

FBGa (mmol/L) – 5.6 (5.2, 5.9) 12.5 (11.0, 12.9) \0.001*

HbA1ca (%) – – 10.6 (10.0, 11.1)

Period of ulcerationa (day) – 90.0 (75.0, 100.0) 75.1 (60.0, 83.0) 0.183

a Data are presented by the median and inter-quartile rangeb Data are presented by count and percentage

* Indicates a significant difference in FBG between NDU and DU groups

Acta Diabetol

123

50-ccaaaccggtgactttctgt-30 (150 bp); OB-RL: sense, 50-tactttggaagcccctgatg-30 and antisense 50-gctcaaacgtttctggctt

c-30 (705 bp); and b–actin: sense, 50-agagctacgagctgcctga

c-30 and antisense, 50-aaagccatgccaatctcatc-30 (499 bp).

After preparing the cDNA, the PCR reaction mixture

was composed of the following: 2.0 lL reaction buffer, 0.4

lL 10 mM dNTP, 10.4 lL sense primer 10 pmol/u, 10.4

lL antisense primer 10 pmol/u, 1.8 lL cDNA, 0.3 lL Taq

enzyme, and 13.5 lL ddH2O. The following cycles were

used for the PCR: one cycle at 94 �C for 2 min, 55 �C for

1 min, and 72 �C for 2 min, 30 cycles at 94 �C for 45 s,

55 �C for 40 s, and 72 �C for 1 min, and one cycle at

72 �C for 10 min and 4 �C for 5 min. The mixture was

stored at -94 �C until further analysis.

Agarose gel electrophoresis was performed on the PCR

products followed by image analysis using the Image Tool

3.0 to calculate the relative gene expression values. The

relative gene expression value (%) was determined using

the following equation = [Band area of the target gene 9

(Band luminance - Background luminance)] 7 [Band area

of the b-actin gene 9 (Band luminance - Background

luminance)] 9 100.

Statistical analysis

The continuous data are presented as median and inter-

quartile ranges (i.e., the range between the 25th and

75th percentiles). The nonparametric Kruskal–Wallis and

Mann–Whitney tests were performed for the comparisons

between three and two groups, respectively. The categori-

cal data and gender are presented as counts and percent-

ages. The Fisher’s exact test was performed for the

comparison of gender distribution between groups. The

Spearman’s correlation coefficient (q) was performed to

show the correlation between the period of ulceration and

the expression level of leptin and OB-RL. All statistical

analyses were two sided with the statistical significance

level of 0.05. All statistical assessments were performed

using the SPSS 15.0 statistics software (SPSS Inc, Chicago,

IL, USA).

Results

Patient characteristics

A total of 30 samples were investigated. As shown in

Table 1, no significant differences in age and gender were

observed between the groups. FBG in DU group was sig-

nificantly higher than in NDU group (P \ 0.001). Fur-

thermore, the period of ulceration was also comparable in

the NDU and DU groups.

Tissue morphology

Histological changes, including increased cuticle thicken-

ing, absence of the granular layer, and disordered epider-

mis and prickle cell layer, were observed in the tissue

samples obtained from both the NDU and DU groups

(Fig. 1a). Specifically, significantly thicker cuticles were

observed in the NDU and DU groups as compared to the

NC group (1.80 and 1.67 mm vs. 1.02 mm, respectively;

P \ 0.001; Fig. 1b). In contrast, the hypodermal layer was

significantly thinner in the NDU and DU groups as com-

pared to the NC samples (1.45 and 1.40 mm vs. 2.73 mm,

respectively; P \ 0.001; Fig. 1b). Moreover, disorderly

fibroblasts and collagen bundles, as well as infiltration of

inflammatory cells, were observed in both the NDU and

DU groups.

Expression of leptin and OB-RL

As determined using immunohistochemistry, leptin and

OB-RL expressions were analyzed in all three groups

(Fig. 2a). As compared to the NC group, the median per-

centage of cells with leptin expression was significantly

greater in the NDU and DU groups (36.7 vs. 71.0 and

59.0 %, respectively; P \ 0.001; Fig. 2b). No significant

difference in leptin expression was observed between the

NDU and DU groups. Whereas OB-RL expression was

significantly increased in the NDU group, it was signifi-

cantly decreased in the DU group as compared to the NC

group (73.9 and 21.1 vs. 37.0 %, respectively; P \ 0.001;

Fig. 2b).

Within NC tissue, OB-RL expression was detected pri-

marily in the cytoplasm of basal cells (Fig. 3a), cells of the

sweat gland (Fig. 3b), endothelial cells (Fig. 3c), glandular

sebaceous cells (Fig. 3d), and fibroblasts (Fig. 3e). How-

ever, within DU ulcers, OB-RL protein expression was

rarely observed within keratinocytes, fibroblasts, and

inflammatory cells; leptin protein expression was observed

primarily in the cytoplasm of the keratinocytes and in

some fibroblasts, and macrophages (Fig. 4). In the NDU

group, strongly positive OB-RL expression was obser-

ved primarily in the cell membrane and cytoplasm

of keratinocytes, fibroblasts, and marcrophages; leptin

expression was observed in the cytoplasm of these cells

(Fig. 4).

To determine whether the differential leptin and OB-RL

expression was due to altered gene expression, leptin and

OB-RL mRNA expressions were measured in both the NC

and DU groups. Significantly reduced leptin and OB-RL

mRNA expression was observed in the DU group as

compared to that of NC group (Leptin: 0.23 vs. 0.47,

P = 0.00001, OB-RL: 0.29 vs. 0.44, P = 0.00002;

Fig. 5).

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Correlation of OB-RL expression with the period

of ulceration

To determine whether leptin or OB-RL expression was

associated with patient characteristics, Spearman’s corre-

lation analysis was undertaken. Although no significant

association was observed between patient characteristics

and leptin levels, the period of ulceration was negatively

correlated with OB-RL protein expression in DU group

(q = -0.671, P = 0.034). (Table 2).

Discussion

Because previous studies have proposed that low leptin

levels are associated with poor wound healing, this study

aimed to investigate the relationship between leptin and

OB-RL expression in DFU. In the DU and NDU groups,

significantly greater leptin protein was observed as com-

pared to the NC group. OB-RL protein and mRNA

expression was significantly lower in the DU group; its

protein expression was significantly higher in the NDU

group. Finally, ulcer duration in the DU group was nega-

tively correlated with OB-RL expression.

Leptin is a polypeptide secreted by adipose tissue [16],

regulating appetite and energy consumption [17]. It is also

intimately associated with diabetes, serving as an inde-

pendent marker of bone mineral density [18], as well as

correlating with fasting insulin sensitivity [19] in patients

with type 2 diabetes. A role of leptin in the wound-healing

process has also been suggested [20–25]. Specifically, it

Fig. 1 Ulcerative tissue

organization. a Histological

examination of normal skin (toppanels), chronic diabetic ulcers

(middle panels), and chronic

non-diabetic ulcers (bottompanels), magnification 9100

(left panels). Epidermal (E) and

dermal (D) tissues with greater

magnification, 9200. b Cuticle

and hypodermal layer thickness

among the three groups.* indicates a significant

difference as compared to the

NC group; P \ 0.001

Acta Diabetol

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regulates inflammation, activates macrophages, promotes

angiogenesis, stimulates fibroblast proliferation and colla-

gen synthesis, and accelerates re-epithelialization [26–31].

In addition, leptin is associated with inflammatory marker,

such as C-reactive protein [32] and enhances mitochondrial

activity and biogenesis, which may further affect wound

healing [8]. In the present study, although leptin levels

were not associated with time to wound healing, OB-RL

Fig. 2 Leptin and OB-RL

protein expressions in normal

and ulcerative tissues. a Leptin

(left panels) and OB-RL (rightpanels) expressions were

examined using

immunohistochemistry in

normal (top panels), DU

(middle panels), and NDU

(bottom panels) tissues,

magnification 9100.

b Comparison of leptin and OB-

RL protein expression levels

between the three groups.*indicates a significant

difference as compared to NC

group. � indicates a significant

difference as compared to NDU

group; P \ 0.001

Acta Diabetol

123

protein expression was negatively associated with period of

ulceration, suggesting an association between altered leptin

signaling and delayed wound healing.

In normal skin samples, leptin and OB-RL were widely

observed in cells of the basal layer, fibroblasts, sebaceous

gland cells, sweat gland cells, and vascular endothelial

cells, suggesting that leptin participates in the normal

physiology of mature cutaneous tissues. However, its role

in sweat and sebaceous gland cells remains unclear. In

contrast, greater leptin expression was observed in the

marginal dermal tissues of both diabetic and non-diabetic

foot ulcers in the present study without differences in cell-

type expression, suggesting that prolonged healing in DU

and NDU is not due to reduced leptin expression. Simi-

larly, changes in the leptin receptor, OB-RL, over time

have been reported, with an initial downregulation that

subsequently increased 5 days post-wounding [33]. Thus,

leptin may act as an upstream regulator of cytokine

Fig. 3 The expression of OB-RL in normal skin. OB-RL protein expression was determined using immunohistochemistry; it was detected in

(a) basal cells, b sweat gland cells, c endothelial cells, d sebaceous gland cells, and e fibroblasts, magnification 9400

Acta Diabetol

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expression in normal wound healing, and altered leptin

levels may result in aberrant expression of other factors

that regulate the wound-healing process [34].

Whereas increased leptin protein expression was

observed in the DU group, significantly decreased OB-RL

expression was detected as compared to the normal control

skin. This finding is in accordance with the study by Goren

et al. [30]. In contrast, the expression of OB-RL in the

marginal dermal tissues of the NDU group was higher than

those of the NC group despite having similar levels of

leptin as the DU group, suggesting different underlying

mechanisms. In the DU group, the low OB-RL expression

may possibly be due to resistance or insensitivity of dermal

tissue toward leptin, resulting in the inability of leptin to

exert its regulatory effects on healing. Thus, altered leptin

signaling may be a significant factor accounting for

reduced healing in DFUs, which is in agreement with a

previous study [30]. In a study by Dihn et al. [35], both

diabetic subjects at low and high risk for developing DFUs,

as determined by their neuropathic status, had increased

leptin levels as compared to normal control subjects. Fur-

thermore, upregulation of protein tyrosine phosphatase 1B

Fig. 4 Expression of OB-RL protein in DU and NDU ulcers as determined by immunohistochemistry. magnification, 9200/400

Acta Diabetol

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(PTP1B), a protein that negatively regulates leptin signal-

ing, was also observed and may account for altered leptin

signaling observed in the present study [35]. Unfortunately,

PTP1B expression in non-diabetic foot ulcers has yet to be

evaluated apparently. Reduced OB-RL expression may

also account for the increased leptin expression observed in

the DU group as a way to compensate for the disrupted

signaling. However, Goren et al. [30] observed an absence

of OB-RL in DFUs, which might be related to the distance

the sample taken from the center of the ulcers, as well as

stimulation of cytokine expression by wound cleansing.

For those with non-diabetic foot ulcers, we speculate that

increased leptin signaling may be a major factor underlying

the faster healing as compared to that observed for DFUs

with all other treatments being the same [30].

Fibroblasts within the wound surface secrete leptin, and

various cells express its receptor [2, 31, 36]. In this study,

Fig. 5 Leptin and OB-RL

mRNA expressions in normal

skin and diabetic ulcers. mRNA

expression was determined

using RT-PCR.

a Representative agarose gel

electrophoresis image of

amplified products.

b Comparison of leptin and OB-

RL mRNA expressions between

the NC and DU groups.*indicates a significant

difference as compared to NC

group; P \ 0.05)

Table 2 The Spearman correlation coefficients (q) in ulcer time vs.

the mRNA and protein expressions of leptin and OB-RL in the DU

group

q P value

Protein expression of Leptin (percentage) 0.224 0.534

Protein expression of OB-RL (percentage) -0.671 0.034*

mRNA expression of Leptin 0.307 0.387

mRNA expression of OB-RL -0.313 0.379

* Indicates the corresponding Spearman correlation coefficients (q)

obtained to statistical significance level

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leptin and OB-RL mRNA expressions in the marginal

dermal tissues of DFUs were lower than that observed in

normal skin. This result is inconsistent with the increased

leptin protein levels observed in the DU group by immu-

nohistochemistry. To avoid the interference of subcutane-

ous fat, a major source of leptin, the adipose layer was

removed immediately after the samples were obtained.

Therefore, the high levels of leptin protein observed in the

marginal dermal tissues of diabetic foot ulcers may have

been secreted from the surrounding adipose tissues, which

would account for the discrepant results observed for leptin

protein versus mRNA expression in the DU group as

compared to the NC group. Also, decreased OB-RL

expression might be related to the negative feedback

resulting from high leptin protein levels.

The limited availability of tissue in this study represents

a limitation that prevented a more in-depth investigation of

the role of leptin and OB-RL in the healing process. Fur-

thermore, NDU samples were obtained from preserved

tissues, preventing the analysis of mRNA expression. Thus,

the role of leptin mRNA expression in non-diabetic foot

ulcers was not explored but will be the subject of further

studies. Finally, the mechanism by which OB-RL is neg-

atively associated with the period of ulceration was not

evaluated in the present study.

In conclusion, elevated leptin and decreased OB-RL

were observed in DFU tissue, suggesting that altered leptin

signaling may be related to the delayed wound healing that

is characteristic of DFUs. Further studies are necessary to

determine the possible benefits of modifying DFU man-

agement by application of topical leptin or its mimetics or

upregulating OB-RL receptor expression.

Conflict of interest None.

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