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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: [email protected]
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).
Acta Diabetol
123
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
123
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
123
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
123
(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
Acta Diabetol
123
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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