- Email: [email protected]
Exendin-4 partly ameliorates - hyperglycemia-mediated tissue damage in lungs of streptozotocin-induced diabetic mice
Accepted Manuscript Title: Exendin-4 partly ameliorates of hyperglycemia-mediated tissue damage in lungs of streptozotocin-induced diabetic mice Authors: Fusun Oztay, Serap Sancar Bas, Selda Gezginci-Oktayoglu, Merve Ercin, Sehnaz Bolkent PII: DOI: Reference:
S0196-9781(17)30376-5 https://doi.org/10.1016/j.peptides.2017.12.007 PEP 69885
To appear in:
Peptides
Received date: Revised date: Accepted date:
17-7-2017 4-12-2017 6-12-2017
Please cite this article as: Oztay Fusun, Bas Serap Sancar, Gezginci-Oktayoglu Selda, Ercin Merve, Bolkent Sehnaz.Exendin-4 partly ameliorates of hyperglycemiamediated tissue damage in lungs of streptozotocin-induced diabetic mice.Peptides https://doi.org/10.1016/j.peptides.2017.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TITLE PAGE
Title. Exendin-4 partly ameliorates of hyperglycemia-mediated tissue damage in lungs of streptozotocin-induced diabetic
SC RI PT
mice
Author names and affiliations.
Fusun Oztay, Serap Sancar Bas, Selda Gezginci-Oktayoglu, Merve Ercin, Sehnaz Bolkent
U
Fusun Oztay, MSc, PhD, Prof.
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134,
A M
Serap Sancar Bas, MSc, PhD, Assistant Prof.
N
ISTANBUL/TURKEY, e -mail: [email protected]
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134,
TE
D
ISTANBUL/TURKEY, e -mail: [email protected]
EP
Selda Gezginci-Oktayoglu, MSc, PhD, Assoc. Prof. Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134,
CC
ISTANBUL/TURKEY, e -mail: [email protected]
A
Merve Ercin, MSc
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134, ISTANBUL/TURKEY, e -mail: [email protected]
Sehnaz Bolkent MSc, PhD, Prof.Dr.
1
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134, ISTANBUL/TURKEY, e-mail: [email protected]
Corresponding author.
SC RI PT
Selda Gezginci-Oktayoglu, MSc, PhD, Assoc. Prof. Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134, ISTANBUL/TURKEY, e -mail: [email protected] Phone:+90 212 455 57 00 (external, 15084)
U
Fax: +90 212 528 05 27
A
CC
EP
TE
D
M
A
N
Graphical abstract
2
Highlights
Exendin-4 contributed to the healing of lung against hyperglycemiamediated increased oxidative stress, tissue damage and pulmonary edema through its glucose-lowering and oxidative stress-reducing effects.
SC RI PT
Exendin-4 induced the cell proliferation in pulmonary epithelium and interstitium.
Exendin-4 results in the disruption of insulin signaling and increases at the protein level of collagen-type-I in the lung of diabetic mice.
N
U
Exendin-4-mediated a critical reduction in the amount of fibronectin may result in excessive collagen accumulation in the lung of diabetic mice.
A
ABSTRACT
M
Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion, and plays anti-inflammatory role in atherosclerosis, and has surfactant-releasing effects in lungs. GLP-1 analogues are used in diabetes therapy. This is the first study to investigate the effects of exendin-4, a GLP-1 receptor agonist, on lung injury in diabetic mice. BALB/c male mice
D
were divided into four groups. The first group was given only citrate buffer, the second group was given only
TE
exendin-4, the third group was given only streptozotocin (STZ), and the fourth group was given both exendin-4 and STZ. Exendin-4 (3 µg/kg) was administered daily by subcutaneous injection for 30 days after mice were rendered diabetic with a single dose of STZ (200 mg/kg). Structural alterations, oxidative stress, apoptosis, insulin signaling
EP
and expressions of prosurfactant-C, alpha-smooth muscle actin, collagen-I and fibronectin were evaluated in lung tissue. Diabetic mice lungs were characterized by induced oxidative stress, apoptosis, edema, and cell proliferation.
CC
They had honeycomb-like alveoli, thick alveolar walls, and hypertrophic pneumocytes. Although exendin-4 treatment improved pulmonary edema, apoptosis, oxidative stress, and lung injury, it led to the disrupted insulin signaling and interstitial collagen accumulation in the lungs of diabetic mice. Exendin-4 reduced hyperglycemia-
A
mediated lung damage by reducing glucose, and oxidative stress and stimulating cell proliferation. However, exendin-4 led to increased risk by reducing insulin signaling in the lungs and collagen accumulation around pulmonary vasculature in diabetic mice.
Keywords: Exendin-4, diabetes, lung injury, insulin resistance, fibrosis.
3
1. Introduction
Type 2 diabetes, obesity and metabolic syndrome are common comorbidities of some chronic lung diseases [40].
SC RI PT
Clinical and experimental studies on diabetic animals and patients have reported a reduction in lung elastic recoil, lung-diffusing capacity, and pulmonary capillary blood volume, as well as induced pulmonary oxidative stress,
structural damage of alveolar areas, and thicker alveolar basement membranes [33, 35]. The disruption of insulin signaling is a common feature of the metabolic diseases mentioned above and it is considered to be a strong risk
factor for lung diseases [37]. Type 2 diabetes is a metabolic disease characterized by insulin resistance, which arises from impaired insulin signaling in insulin-sensitive cells. Insulin signaling is triggered by the insulin receptor, which is a kinase that phosphorylates the tyrosine residues of insulin receptor substrate (IRS) that in turn activates AKT
U
with the phosphorylation of its serine residues. Insulin resistance develops as a result of long-term high-level insulin levels in the blood and is characterized primarily by an increase in phospho (p)-IRS1 (ser307) and a decrease in p-
N
AKT (ser473) in cells [5]. Although the lack of cytokine signaling-3 (SOCS3) is generally considered to play a negative
A
role in this mechanism [5], there is also evidence that insulin resistance progresses in the absence of this molecule [41]. The presence of insulin receptors in the lungs was demonstrated years ago [43]. The negative effects of
M
gestational diabetes on fetal lung development are related to increased maternal insulin levels [2]. In addition, disrupted insulin signaling has been reported to negatively affect lung function in adolescents [17]. Moreover, high
D
levels of insulin have been reported to induce contraction in the airway smooth muscle [34], and insulin increases the extracellular matrix proteins by stimulating the proliferation and contraction of the airway smooth muscle [9].
TE
Hence, diabetes may contribute to pulmonary complications but, studies on the molecular mechanism of lung
EP
dysfunction in diabetics are insufficient.
Glucagon-like peptide-1 (GLP-1) stimulates mainly glucose-induced insulin secretion and reduces glucagon
CC
release [23, 30]. GLP-1 and its analogues can increase beta-cell mass in animal models with type 2 diabetes and human islet cells [19, 42]. Exendin-4, a GLP-1 receptor agonist, regulates insulin production and glycemic control in
A
diabetes [20]. Exendin increases insulin secretion [10]. For these reasons, exendin-4 has been used to treat type 2 diabetic patients. GLP-1 releases surfactant in the lungs [44]. GLP-1 receptors have reduced mortality and improved lung function in a model of experimental obstructive lung disease in female mice [45]. However, the therapeutic effect of GLP-1 and its receptor agonists on diabetes-mediated lung damage is unclear. The present study examines the therapeutic effect of exendin-4, which has similar functional properties to native GLP-1, on lung injury in diabetic mice. The study evaluates lung tissue for the following: structural alterations, oxidative stress, apoptosis, and insulin signaling as well as expressions of prosurfactant-C (pro-SPC, type II pneumocyte marker), alpha-smooth
4
muscle actin (α-SMA, myofibroblast marker) collagen-I, fibronectin, and insulin signaling regulators (p-IRS1, p-AKT and SOCS3).
2. Materials and methods
SC RI PT
2.1. Animals and experimental design All experiments were performed in accordance to the guidelines of the Istanbul University Local Ethic Committee of Experimental Animals. The mice were provided from Experimental Medical Research Institute of Istanbul
University. They were kept at a stable temperature (22 ± 1ºC) for 12 h light and dark cycles and fed with a standard pellet chow.
BALB/c male mice at 10 weeks of age were divided into four groups (10 animals per group). The first group was given citrate buffer only, the second group was administered exendin-4 alone, the third group received
U
streptozotocin (STZ), and the fourth group was given both STZ and exendin-4. The exendin-4 (Sigma, St. Louis, MO,
N
3 μg/kg dissolved in ultrapure water) was daily administered by subcutaneous injection for 30 days after mice were rendered diabetic by administration of a single dose of STZ (Sigma, St. Louis, MO, 200 mg/kg dissolved in 0.01 M
A
citrate buffer, pH: 4.5). Vehicle or exendin-4 was injected to control animals once every 24 hours. The animals were
M
fasted overnight prior to the experiments, but they were allowed to drink water. On day 30, the animals were
TE
2.2. Histopathological evaluation
D
sacrificed by cervical dislocation and the lung tissues were taken immediately.
Pieces of lung tissue were fixed in Bouin’s solution. The tissues were washed with 70% ethanol to remove picric acid, dehydrated in graded ethanol series and embedded in paraffin wax. Section of 4 µm thickness were taken
EP
serially and stained with Masson’s trichrome. Microscopic analysis of tissues was made using a light microscope (Olympus, CX41) and the photomicrographs were taken with Olympus DP71 digital camera. The lung injury was
CC
evaluated in the lung sections according to the following criteria: disintegrity of pulmonary epithelium, structural alterations in alveolar areas and bronchioles, the accumulation of collagen in the connective tissue, and the
A
presence of the pulmonary inflammation and edema.
2.3. Immunohistochemistry Sections of lungs, mounted on poly-L-lysine slides, were cleared in toluene, hydrated through decreasing ethyl alcohol series and boiled in 10 mM citrate buffer (pH 6), [27]. After incubation in 3% H2O2 in methanol and 0.3 %Triton-X 100, sections were incubated with anti-proliferative cell nuclear antigen (PCNA) antibody for 30 min at
5
room temperature or anti-cleaved caspase-3 antibody at 4°C overnight. The antibodies used are listed in Table 1. The immunostaining was carried out by using the Histostain Plus Kit (Invitrogen, Paisley, Scotland) comprising the streptavidin-biotin-horseradish peroxidase method, and revealed using 3-amino-9-ethylcarbazole as chromogen. Percentage of stained cells was quantified by following formula: (Ʃ immunopositive cell / Ʃ cell) x 100.
SC RI PT
2.4. Western blotting
The lungs were homogenized in cold RIPA buffer. The homogenates were centrifuged for 10 minutes at 4°C and 10.000g. The supernatant was removed for electrophoresis. Equal amounts of protein (80 μg) were separated on SDS-PAGE gels (7.5%, 10% and 12%) and transferred to nitrocellulose membrane. The blots were blocked for nonspecific binding for 1 hour at room temperature in 5% fat-free dry milk in TBST. Thereafter the blots were
incubated at 4°C with primary antibodies overnight. The antibodies are listed in Table 1. After washing, specific
U
bands were visualized with the respective horseradish peroxidase-conjugated secondary antibodies (Table1) and
M
2.5. Measurement of oxidative stress
A
Imaging Software and normalized to β-actin protein bands.
N
Luminol reagent (Santa Cruz, CA, USA). The intensities of protein bands were quantified using the Kodak Molecular
Tissue reactive oxygen species (ROS) generation was measured according to the method of Lebel and Bondy
D
[24] further modified by Kim et al [22]. ROS production was measured by the oxidation of a poorly fluorescent non-
TE
ionic, non-polar reagent 2',7'-dichlorofluorescein diacetate (DCFH-DA) into the highly fluorescent 2',7'dichlorofluorescein. Briefly, lung tissue homogenates containing 50 µg protein were transferred to black 96-well microplates and taken to 100 μl with assay medium (20 mM Tris -HCl, 130 mM KCl, 5 mM MgCl2, 20 mM NaH2PO4,
EP
30 mM glucose). DCFH-DA (10 µg/ml, 10 µl volume) dissolved in dimethylsulfoxide, was added to the mixture in the dark. After incubation at 37ºC for 3h, fluorescent product was measured at 485 nm excitation and 525 nm emission
CC
wavelength by a spectrofluorometer. Then, ROS generation was calculated as the % of control. In addition, oxidative damage, was determinated by measuring malondialdehyde (MDA) levels according to the protocol described by Ledwozwy and coworkers [25].
A
2.6. Statistical analysis Data are expressed as mean ± S.E.M. The results were analyzed by one-way ANOVA followed by Tukey’s post
hoc test to compare differences among groups by using GraphPad Prism software, version 5.00 (San Diego, CA).
3. Results
6
3.1. Exendin-4 treatment reduced lung injury in diabetic mice Fasting blood glucose values were measured with a glucometer on day 0 and day 30 of the experiment, and individuals with values above 126 mg/dL were considered diabetic according to a previous report [6]. The fasting blood glucose concentration was 99.1 ± 2.3 mg/dL in control mice, 92.3 ± 1.6 mg/dL in exendin-4 treated mice,
SC RI PT
380.7 ± 15.7 mg/dL in STZ-injected mice, and 115.1 ± 3.19 mg/dL in exendin-4 injected diabetic mice at day 30 [20]. These results indicate that administration of STZ causes the development of advanced diabetes at the indicated dose and time.
Diabetes induced the disruption of alveolar structure. Also, diabetic mice had honeycomb-like alveoli, thick alveolar walls and hypertrophic pneumocytes in their lungs. It was detected fluid accumulation in the tissue.
U
Exendin-4 treatments resulted in the regression of edema and healing of alveolar structure. However, increase in
N
cell number around bronchioles and vessels, and interstitial collagen accumulation were evident in diabetic mice
M
A
treated with exendin-4 (Fig. 1).
3.2. Exendin-4 treatment reduced oxidative stress seen in the diabetic lungs.
D
The total ROS levels in the lung tissues from diabetic animals were higher than the control group (p < 0.05), and treatment of diabetic mice with exendin-4 normalized ROS levels (p < 0.05) (Fig. 2A). On the other hand,
TE
exendin-4 and STZ-induced diabetes caused an increase in the levels of MDA versus control group (p < 0.05, p < 0.001 respectively). Treatment of diabetic mice with Exendin-4 ameliorated MDA levels only in the Exendin-4
EP
injected group (p < 0.01) (Fig. 2B).
CC
3.3. Exendin-4 treatment reduced apoptotic cells in the diabetic lungs The levels of active caspase-3 were higher in lung tissue in diabetic group versus the control group (p < 0.001)
and exendin-4 reduced the levels of this active enzyme versus diabetic group (p < 0.01) (Figs. 3A and B). In
A
agreement with these results, the apoptotic cells were identified as pulmonary epithelial and interstitial cells in alveolar areas of diabetic mice under light microscope and the number of these cells was reduced with exendin-4 treatment (Fig. 3C). 3.4. Exendin-4 treatment increased the levels of pro-SPC and RAGE proteins in the diabetic lungs Diabetes did not significant alter the levels of pro-SPC and receptor of advanced glycation end-products proteins (RAGE, a marker of type I pneumocyte) in the lung (p > 0.05) (Figs. 4A and B). However, exendin-4
7
treatment increased the levels of these proteins in diabetic mice (p < 0.001 and p < 0.01, respectively) (Figs. 4A and B).
3.5. Exendin-4 increased cell proliferation in the diabetic lungs
SC RI PT
There was not any significant changes in the number of PCNA-immunoreactive (PCNA+) cells in diabetic animals versus control group (p > 0.05). However, exendin-4 treatment to diabetic group caused an increase in PCNA+ cell number versus diabetic mice (p < 0.05), exendin-4 injected control mice (p < 0.05) and vehicle-injected control mice (p < 0.01). Increase in PCNA+ cell number around bronchioles and venules was observed in diabetic mice treated with exendin-4 (Figs. 5A and B).
U
3.6. Exendin-4 induced the production and accumulation of collagen-I in the diabetic lungs
N
Diabetic mice were characterized by the decreased α-SMA (p < 0.01) and active TGFβ-1 (p < 0.05) levels and the increased fibronectin levels (p < 0.05) versus control group (Figs. 6A-E). The significant increase in the levels of
A
collagen-I protein could not be determined in the lungs of diabetic mice (Figs. 6A-E). Exendin-4 increased the levels
M
of collagen-I proteins (p <0.01) and α-SMA proteins in the lung of diabetic mice (Figs. 6A-C). Exendin-4-mediated collagen-I production was confirmed by microscopic finding such as interstitial collagen accumulation around pulmonary vessels. However, fibronectin (p < 0.05) and active TGFβ-1 (p < 0.05) levels were decreased by exendin-4
TE
D
treatments in the lung tissue of diabetic mice (Figs. 6D and E).
EP
3.7. Exendin-4 reduced insulin signaling in the diabetic lungs We did not observe the disrupted insulin signaling, which can occur in absence of alteration in p-IRS1 (p > 0.05), SOCS3 (p < 0.001) and p-AKT levels in the lung of diabetic group in comparison with the control group.
CC
However, exendin-4 treatment in diabetic mice caused an increase in p-IRS1 (p < 0.05), decrease in SOCS3 (p<0.001) and p-AKT levels (p < 0.05) in comparison with the diabetic group, indicating the disruption of insulin signaling. In contrast, exendin-4 treatment in control mice resulted in a decrease in p-IRS1 level (p < 0.05)
A
compared to the control group, which suggested that exendin-4 may reduce insulin signaling in lung only under hyperglycemic condition (Figs. 7A-C).
4. Discussion
8
Diabetes-induced lung damage is generally disregarded by patients and physicians, because it is subclinical. However, studies on the lungs of diabetic animals have shown enlargement of air sacs, alveolar wall thickening, structural damage of alveoli, intense inflammatory reactions, and increased pulmonary edema as well as reductions in surfactant proteins and lung-diffusing capacity [1, 13, 28, 33]. Diabetic lungs are characterized by the overproduction of ROS, reactive nitrogen species, secondary ends of lipid peroxidation such as MDA and a decrease
SC RI PT
in the capacity of the pulmonary antioxidant defense system and oxidative stress [16, 26]. Xiong et al. [48] reported elevated MDA levels in serum of diabetic mice. In the lungs they noted hyperglycemia-induced inflammatory
mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 and interleukin-10. An increase in oxidants and a decreased in pulmonary antioxidant enzymes lead to increased cellular stress and consequently, structural damage develops in diabetic lungs. In the present study, hyperglycemia led to similar structural damage and alterations of oxidative stress parameters in diabetic mice lungs. Our study and others, suggest that increased ROS and MDA
levels, and induced structural lung damage are hallmarks of hyperglycemia-mediated alterations in diabetic mice
U
lungs. It is well known that exendin-4 regulates insulin production and glycemic control in diabetes [10, 20].
Gezginci-Oktayoglu and Bolkent [20] showed that exendin-4 increased β-cell counts and insulin production in mice.
N
Exendin-4 administered to diabetic mice significantly reduced blood glucose, the levels of interleukin-1β and TNF-α,
A
and oxidative stress in kidneys [36]. Similarly, in the present study focused on diabetic mice lungs, regression of hyperglycemia via exendin-4 administration attenuated hyperglycemia-mediated abnormal structural alterations
D
M
and oxidative stress.
Chronically high glucose uptake can lead to glucotoxicity and cell death. When high glucose is consumed by
TE
the mitochondrial electron transport system, ROS production increases. Proteins released from mitochondria into the cytosol and the subsequent caspase-3 activation promote cell apoptosis [38]. In diabetic rat lungs, high glucose induces apoptosis through redox imbalance, mitochondrial abnormalities, and caspase-3 activation [47]. These data
EP
are confirmed by several findings in the present study such as the presence of many cleaved-caspase-3+ cells in the pulmonary epithelium and interstitium and increases in cleaved-caspase-3 protein, ROS, and MDA in lung tissue.
CC
Thus, the present study supplies valuable data on hyperglycemia-mediated damage to the lungs. The pulmonary epithelium covering the inner surface of the alveoli is very sensitive to oxidative stress. It consists of two types of epithelial cells, type I and II pneumocytes. Type II pneumocytes, which produce surfactant proteins, play a role in
A
regenerating pulmonary epithelium by supporting its survival after injury. They proliferate and subsequently differentiate into pulmonary epithelium cells as a response to tissue damage [14]. In agreement with this the present study shows pulmonary epithelial cell hyperplasia, increased cleaved-caspase-3+ and PCNA+ cells in the pulmonary epithelium and interstitium which are likely signs of diabetes-mediated tissue damage. We postulate that diabetes induces cell apoptosis and proliferation in both pulmonary epithelium and interstitium in mice lungs as a response to tissue damage. On the other hand, the pulmonary epithelium is close to pulmonary capillaries. Cell
9
junctions between pulmonary epithelial cells prevent accumulation of fluids in alveolar areas. Diabetic complications in lung tissue are related to pulmonary epithelial and endothelial dysfunctions. Diabetes-induced hyperglycemia and increased oxidative stress cause epithelial cell death and vascular permeability [7]. The present study clearly demonstrates pulmonary edema in diabetic mice lungs. Based on previous studies and the present one we suggest that diabetes-induced pulmonary epithelial damage as well as pulmonary epithelial and endothelial
SC RI PT
dysfunction lead to pulmonary edema. Exendin-4 in diabetic mice ameliorated of hyperglycemia-mediated increased oxidative stress, tissue damage and pulmonary edema in lung tissue because it reduces glucose and
oxidative stress. Diabetic mice who received exendin-4 had fewer cleaved-caspase-3+ cells, reduced levels of ROS and MDA, more PCNA+ cells, and RAGE and pro-SPC proteins. RAGE and pro-SPC proteins are cell markers of type I and II pneumocytes, respectively. Hence, the increases in RAGE and pro-SPC proteins interpreted as repair of the
pulmonary epithelium. Thus, we suggest that exendin-4 contributes to the repair of the pulmonary epithelium and interstitium by stimulating cell proliferation. A proliferative effect of exendin-4 was also reported by Baggio and
U
Drucker [3]. They noted that exendin-4 induced pancreatic β-cell proliferation in rodents and ameliorated glucose
A
preosteoblasts via activation of the MAPK pathway [15].
N
metabolism in diabetic animals and patients. It was also shown that exendin-4 promoted cell proliferation of
M
In diabetes extracellular matrix components accumulate and the basement membrane thickens in the lungs and kidneys [18, 46]. As a complication of diabetes, fibrosis can occur in these tissues as well as diabetic
D
nephropathy and retinopathy [4]. Tissue fibrosis is characterized by an increase fibroblasts and myofibroblasts expressing α-SMA and an accumulation of extracellular matrix components such as collagens, fibronectin,
TE
proteoglycans, and hyaluronic acid in the matrix [32, 49]. This accumulation and excessive production are modulated by several growth factors and TGF-β [8]. Enomoto et al. [12] reported that diabetes may be a risk factor for idiopathic pulmonary fibrosis. However, instead of a fibrotic response, thickening basement membranes
EP
were observed in diabetic lungs. Zheng et al. [50] detected basement membrane thickening of the alveolar wall through excessive production and accumulation of collagen-IV. In the present study, diabetic mice that did not
CC
receive exendin-4 did not exhibit fibrotic alterations in the lungs. They did not have increased levels of activatedTGF-β (a profibrotic cytokine), α-SMA and collagen-I proteins, except for fibronectin. In contrast, mice that did receive exendin-4 showed greater production and accumulation of collagen-I protein around the pulmonary
A
vasculature. These mice showed lower levels of activated-TGFβ and fibronectin, which is present in these areas. Although TGF-β is an important profibrotic cytokine, the production and accumulation of extracellular matrix components can be stimulated independently of TGF-β [31]. Kawelke et al. [21] reported that collagen-I production was more pronounced by fibronectin-deficient hepatic stellate cells. They found that removing fibronectin from the liver and hepatic stellate cells led to an increase in baseline collagen-I as a response to induced experimental fibrosis. On the basis of these data, we postulate that fibronectin regulates collagen production. In
10
our study, exendin-4 mediated collagen production and accumulation around the pulmonary vasculature can be related to the reduction of fibronectin in diabetic mice lungs. Furthermore, exendin-4 induced the production and accumulation of collagen by stimulating mesenchymal cell proliferation in diabetic mice. This was confirmed by the presence of PCNA+ cells in areas where interstitial collagen is deposited around the pulmonary vasculature in the
SC RI PT
diabetic mice who received exendin-4.
Lung epithelial cells strongly express insulin receptor in the pseudoglandular stage. In contrast, insulin
receptors decrease in more advanced stages of development [39]. Disruption of insulin signaling has been reported to negatively affect lung function of adolescents [Forno et al., 2015]. This distribution can be tracked with SOCS3, pIRS1, and p-AKT proteins [29]. Insulin leads to the activation of IRS proteins when bound to their receptors, then
these proteins direct many cellular events namely growth and survival in different cell types via PI3K/AKT signaling [Singh et al., 2013]. An increase in serine 307 phosphorylation of IRS1 is the sign of disrupted insulin signaling [5], in
U
contrast, tyrosine phosphorylation of IRS1 leads to insulin signaling. SOCS3 is a regulator of insulin signaling
N
especially induced by cytokines and insulin [11]. In the present study, exendin-4 treatment caused an increase in IRS1 phosphorylation and a decrease in SOCS3, which means that the disruption of insulin signaling occurred in the
A
lungs. Although exendin-4 administration to diabetic mice contributed to lungs healing of tissue damaged by
M
hyperglycemia, the disruption of insulin signaling may have a negative impact on lung biology.
D
6. Conclusion
TE
Diabetes has major impacts on the lungs, where it induces oxidative stress, apoptotic cell death, edema, and disrupts the alveolar structure. Exendin-4 administration to diabetic mice contributes to the healing of lung against hyperglycemia-mediated increased oxidative stress, tissue damage and pulmonary edema by its glucose-lowering
EP
and anti-oxidant effects. Additionally, it induced tissue repair in pulmonary epithelium and interstitium by stimulating cell proliferation. Nevertheless, exendin-4 disrupted insulin signaling and increased protein levels of
CC
collagen-I in the lungs of diabetic mice. Unexpectedly, it decreased fibronectin protein levels. Exendin-4-mediates a critical reduction in the amount of fibronectin that may result in excessive collagen accumulation around the
A
pulmonary vasculature.
Conflict of interest The authors declare that they have no conflict of interest to disclose.
11
Acknowledgements This work was supported by Scientific Research Projects Coordination Unit of Istanbul University with the project number 408. The authors thank to Dr. Anna Cosagliola from Napoli Federico II University for editing of the
SC RI PT
manuscript.
References
[1] Alkan M, Celik A, Bilge M, Kiraz HA, Kip G, Ozer A, et al. The effect of levosimendan on lung damage
after myocardial ischemia reperfusion in rats in which experimental diabetes was induced. J Surg Res 2014;
U
193:920-25.
N
[2] Azad MB, Becker AB, Kozyrskyj AL. Association of maternal diabetes and child asthma. Pediatr Pulmonol
A
2013; 48:545-552.
M
[3] Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007; 132:2131–57.
D
[4] Ban CR, Twigg SM. Fibrosis in diabetes complications: pathogenic mechanisms and circulating and urinary
TE
markers. Vasc Health Risk Manag 2008; 4:575-96.
[5] Boucher J, Kleinridders A, Kahn CR. Insulin receptor signaling in normal and insulin-resistant states. Cold
EP
Spring Harb Perspect Biol 2014; 6(1). pii: a009191. doi: 10.1101/cshperspect. a009191.
CC
[6] Cefula W. Standards of medical care in diabetes-2014: Summary of revisions. Diabetes Care 2014; 38:1-93.
[7] Clemmer JS, Xiang L, Lu S, Mittwede PN, Hester RL. Hyperglycemia-mediated oxidative stress increases
A
pulmonary vascular permeability. Microcirculation 2016; 23:221-9.
[8] Cutroneo KR, White SL, Phan SH, Ehrlich HP. Therapies for bleomycin induced lung fibrosis through regulation of TGF-beta1 induced collagen gene expression. J Cell Physiol 2007; 211:585-9.
[9] Dekkers BG, Schaafsma D, Tran T, Zaagsma J, Meurs H. Insulin-induced laminin expression promotes a hypercontractile airway smooth muscle phenotype. Am J Respir Cell Mol Biol 2009; 41:494-504.
12
[10] Egido EM, Silvestre RA, Hernández R, Marco J. Exendin-4 dose-dependently stimulates somatostatin and insulin secretion in perfused rat pancreas. Horm Metab Res 2004; 36(9):595-600.
[11] Emanuelli B, Peraldi P, Filloux C, Sawka-Verhelle D, Hilton D, Van Obberghen E. SOCS-3 is an insulininduced negative regulator of insulin signaling. J Biol Chem 2000; 275:15985-91.
SC RI PT
[12] Enomoto T, Usuki J, Azuma A, Nakagawa T, Kudoh S. Diabetes mellitus may increase risk for idiopathic pulmonary fibrosis. Chest 2003; 123(6):2007-11.
[13] Eren G, Cukurova Z, Hergunsel O, Demir G, Kucur M, · Uslu E, et al. Protective effect of the nuclear factor kappa B inhibitor pyrrolidine dithiocarbamate in lung injury in rats with streptozotocin-induced diabetes. Respiration 2010; 79: 402–10.
U
[14] Evans MJ, Cabral LJ, Stephens RJ, Freeman G. Renewal of alveolar epithelium in the rat following
N
exposure to NO2. Am J Pathol 1973; 70(2):175-98.
A
[15] Feng Y, Su L, Zhong X, Guohong W, Xiao H, Li Y, Xiu L. Exendin-4 promotes proliferation and
M
differentiation of MC3T3-E1 osteoblasts by MAPKs activation. J Mol Endocrinol 2016; 56:189-99.
[16] Forgiarini LA. Jr Kretzmann NA, Porawski M, Dias AS, Marroni NA. Experimental diabetes mellitus:
D
oxidative stress and changes in lung structure. J Bras Pneumol 2009; 35:788-91.
TE
[17] Forno E, Han YY, Muzumdar RH, Celedón JC. Insulin resistance, metabolic syndrome, and lung function in US adolescents with and without asthma. J Allergy Clin Immunol 2015; 136:304-11.
EP
[18] Friedman E. Diabetic renal disease. In: Rifkin H, Porte D Jr, eds. Ellenberg and Rifkin's Diabetes Mellitus:
CC
Theory and Practice. 4th ed. New York, NY: Elsevier; 1990;684–709.
[19] Garber AJ. Incretin effects on β-cell function, replication, and mass: The human perspective. Diabetes Care
A
2011; 34:258–63.
[20] Gezginci-Oktayoglu S, Bolkent S. Exendin-4 exerts its effects through the NGF/p75NTR system in diabetic mouse pancreas. Biochem Cell Biol 2009; 87:641–51.
[21] Kawelke N, Vasel M, Sens C, Au Av, Dooley S, Nakchbandi IA. Fibronectin protects from excessive liver fibrosis by modulating the availability of and responsiveness of stellate cells to active TGF-β. PLoS One 2011; 6(11):28181.
13
[22] Kim JD, McCarter RJ, Yu BP. Influence of age, exercise, and dietary restriction on oxidative stress in rats. Aging 1996; 8:123-29.
[23] Kolterman OG, Buse JB, Fineman MS, Gaines E, Heintz S, Bicsak TA, et al. Synthetic exendin-4 (AC2993) significantly reduces postprandial and fasting plasma glucose in subjects with type 2 diabetes. J
SC RI PT
Clin Endocrinol Metab 2003; 88:3082–89.
[24] Lebel CP, Bondy SC. Sensitive and rapid quantitation of oxygen reactive species formation in rat synaptosomes. Neurochem Int 1990; 17:435-40.
[25] Ledwozwy A, Michalak J, Stepien A, Kadziolka A. The relationship plasma triglycerides, cholesterol, total
U
lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986; 155: 275-83.
[26] Liang JQ, Ding CH, Ling YL, Xu HB, Lu P, Xian XH. The protective function of puerarin to the injury of
A
N
the lung and its mechanisms during diabetes. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2007; 23:355-8.
[27] Liguori G, Pavone LM, Assisi L, Langella E, Tafuri S, Mirabella N, et al. Expression of orexin B and its
M
receptor 2 in rat testis. Gen Comp Endocrinol 2017; 242:66-73.
TE
Intern Med 2006; 20:47-51.
D
[28] Mexas AM, Hess RS, Hawkins EC, Martin LD. Pulmonary lesions in cats with diabetes mellitus. J Vet
[29] Nicolau J, Lequerré T, Bacquet H, Vittecoq O. Rheumatoid arthritis, insulin resistance and diabetes. Joint
EP
Bone Spine 2016; 1297-319X(16)30153-1.
[30] Nielsen LL, Young AA, Parkes DG. Pharmacology of exenatide (synthetic exendin-4): a potential
CC
therapeutic for improved glycemic control of type 2 diabetes. Regul Pept 2004; 117:77-88.
[31] Oga T, Matsuoka T, Yao C, Nonomura K, Kitaoka S, Sakata D, et al. Prostaglandin F(2alpha) receptor
A
signaling facilitates bleomycin-induced pulmonary fibrosis independently of transforming growth factorbeta. Nat Med 2009; 15:1426-30.
[32] Ovet H, Oztay F. The copper chelator tetrathiomolybdate regressed bleomycin-induced pulmonary fibrosis in mice, by reducing lysyl oxidase expressions. Biol Trace Elem Res 2014; 162:189-99.
14
[33] Oztay F, Kandil A, Gurel E, Ustunova S, Kapucu A, Balci H, et al. Relation between nitric oxide and leptin in the lung of rat with streptozotocin-induced diabetes. Cell Biochem Funct 2007; 26:162–71.
[34] Papagianni M, Hatziefthimiou A, Chachami G, Gourgoulianis K, Molyvdas PA, Paraskeva E. Insulin causes a transient induction of proliferation via activation of the PI3-kinase pathway in airway smooth
SC RI PT
muscle cells. Exp Clin Endocrinol Diabetes 2007; 115:118-23.
[35] Ramirez LC, Dal Nogare A, Hsia C, Arauz C, Butt I, Strowig SM, et al. Relationship between diabetes control and pulmonary function in insulin-dependent diabetes mellitus. Am J Med 1991; 91:371–6.
[36] Sancar-Bas S, Gezginci-Oktayoglu S, Bolkent S. Exendin-4 attenuates renal tubular injury by decreasing oxidative stress and inflammation in streptozotocin-induced diabetic mice. Growth Factors 2015; 33:419-
U
29.
[37] Singh S, Prakash YS, Linneberg A, Agrawal A. Insulin and the lung: connecting asthma and metabolic
A
N
syndrome. J Allergy (Cairo) 2013; 627384.
M
[38] Smart EJ, Li XA. Hyperglycemia: cell death in a cave. Biochim Biophys Acta 2007; 1772:524-6.
[39] Sodoyez-Goffaux FR, Sodoyez JC, De Vos CJ. Insulin receptors in the fetal rat lung. A transient
D
characteristic of fetal cells? Pediatr Res 1981; 15:1303-7.
TE
[40] Song Y, Klevak A, Manson JE, Buring JE, Liu S. Asthma, chronic obstructive pulmonary disease, and type 2 diabetes in the Women’s Health Study. Diabetes Res Clin Pract 2010; 90: 365–71.
EP
[41] Torisu T, Sato N, Yoshiga D, Kobayashi T, Yoshioka T, Mori H, et al. The dual function of hepatic SOCS3
CC
in insulin resistance in vivo. Genes Cells 2007; 12(2):143-54.
[42] Tourrel C, Bailbe´ D, Lacorne M, Meile MJ, Kergoat M, Portha B. Persistent improvement of type 2 diabetes in the Goto– Kakizaki rat model by expansion of the h-cell mass during the prediabetic period with
A
glucagon-like peptide-1 or exendin-4. Diabetes 2002; 51:1443–52.
[43] Ulane RE, Graeber JE, Steinherz R. A comparison of insulin receptors in the developing fetal lung in normal and in streptozotocin-induced diabetic pregnancies. Pediatr Pulmonol 1985; 1:86-90.
[44] Vara E, Arias-Diaz J, Garcia C, Balibrea JL, Blazquez E. Glucagon-like peptide-1(7–36) amide stimulates surfactant secretion in human type II pneumocytes. Am J Respir Crit Care Med 2001; 163:840–6.
15
[45] Viby NE, Isidor MS, Buggeskov KB, Poulsen SS, Hansen JB, Kissow H. Glucagon-like peptide-1 (GLP-1) reduces mortality and improves lung function in a model of experimental obstructive lung disease in female mice. Endocrinology 2013; 154:4503-11.
[46] Watanabe K, Senju S, Toyoshima H, Yoshida M. Thickness of the basement membrane of bronchial
SC RI PT
epithelial cells in lung diseases as determined by transbronchial biopsy. Respir Med 1997; 91(7):406-10.
[47] Wu J, Jin Z, Yan LJ. Redox imbalance and mitochondrial abnormalities in the diabetic lung. Redox Biol 2016; 11:51-9.
[48] Xiong XG, Wang WT, Wand LR, Jin LD, Lin LN. Diabetes increases inflammation and lung injury
U
associated with protective ventilation strategy in mice. Int Immunopharmacol 2012; 13:280–3.
[49] Yilmaz O, Oztay F, Kayalar O. Dasatinib attenuated bleomycin-induced pulmonary fibrosis in mice.
A
N
Growth Factors 2015; 33:366-75.
[50] Zheng F, Lu W, Wu F, Li H, Hu X, Zhang F. Recombinant decorin ameliorates the pulmonary structure
M
alterations by down-regulating transforming growth factor-beta1/SMADS signaling in the diabetic rats.
Figure Legends
TE
D
Endocr Res 2010; 35:35-49.
EP
Fig. 1. Lung histology of mice in the sections stained with Masson’s trichrome. In Exendin-4-treated diabetic mice, collagen fibers accumulated in the extracellular matrix seem to be colored green in the lung sections stained with
CC
Masson’s trichrome. Alveoli (A), terminal bronchiole (TB) and vein (V) honeycomb-like alveoli (□), edema (*) and thick alveolar walls (arrows). CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: diabetic group, and
A
D+Ex-4: Exendin-4-injected diabetic group.
16
SC RI PT U
N
Fig. 2. (A) The graph demonstrates the levels of ROS production (% of CB) per group. CB: 97.12 ± 16.5, Ex-4: 121.1 ±
A
6.95, D: 200.4 ± 20.17, D+Ex-4: 103.0 ± 32.97, *p < 0.05 versus control group; and #p < 0.01 versus diabetic group. (B) The graph demonstrates the levels of MDA (nmol/mg protein) per group. CB: 0.17 ± 0.04, Ex-4: 0.47 ± 0.09, D:
M
0.90 ± 0.06, D+Ex-4: 0.50 ± 0.03, *p < 0.05 versus control group; ***p < 0.001 versus control group, ## p < 0.01 versus diabe c group, ††p < 0.01 versus control group. Values are mean ± S.E.M. n=5 animals for each group. CB:
D
Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-injected diabetic
A
CC
EP
TE
group.
17
SC RI PT U
N
Fig. 3. (A) Western immunoblot of cleaved caspase-3 in lung homogenates. (B) The graph demonstrates the levels
A
of cleaved caspase-3 (arbitrary units) per group. CB: 0.22 ± 0.04, Ex-4: 0.26 ± 0.08, D: 0.82 ± 0.03, D+Ex-4: 0.44 ± 0.11, ***p < 0.001 versus control group; and ##p < 0.01 versus diabetic group. Values are mean ± S.E.M. n=5 animals
M
for each group. (C) Immunopositive reactions of active caspase-3 were shown by arrows for per group. CB: Vehicle-
A
CC
EP
TE
D
injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-injected diabetic group.
18
SC RI PT U N A M D TE EP CC A
Fig. 4. (A) Western immunoblot of pro-SPC and RAGE in lung homogenates. (B) The graph demonstrates the levels of pro-SPC (arbitrary units) per group. CB: 0.67 ± 0.05, Ex-4: 0.86 ± 0.02, D: 0.62 ± 0.01, D+Ex-4: 1.11 ± 0.01, *p < 0.05 and ***p < 0.001 versus control group; ΨΨp < 0.01 versus Ex-4-injected group; and ###p < 0.001 versus diabetic group. (C) The graph demonstrates the levels of RAGE (arbitrary units) per group. CB: 1.01 ± 0.01, Ex-4: 0.96 ± 0.03, D: 1.03 ± 0.01, D+Ex-4: 1.25 ± 0.0, ##p < 0.01 versus diabetic group. Values are mean ± S.E.M. n=5 animals for each
19
group. CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-
A
CC
EP
TE
D
M
A
N
U
SC RI PT
injected diabetic group.
Fig. 5. (A) PCNA+ lung cells (arrows) generally located around the veins (V). (B) The graph demonstrates the percent of PCNA+ lung cells per group. CB: 3.64 ± 0.83, Ex-4: 7.56 ± 1.3, D: 6.75 ± 1.6, D+Ex-4: 13.06 ± 0.62, #p < 0.05 versus diabetic group; **p < 0.01 versus control group. Values are mean ± S.E.M. n=5 animals for each group. CB: Vehicleinjected group, Ex-4: Exendin-4-injected group; D: Diabetic group, and D+Ex-4: Exendin-4-injected diabetic group.
20
SC RI PT U N A M D TE EP CC A
Fig. 6. (A) Western immunoblot of fibrosis markers that α-SMA, collagen-I and fibronectin in lung homogenates. (B) The graph demonstrates the levels of α-SMA (arbitrary units) per group. CB: 1.56 ± 0.05, Ex-4: 1.65 ± 0.11, D: 0.75 ± 0.09, D+Ex-4: 1.24 ± 0.02, **p < 0.01 versus control group; and #p < 0.05 versus diabetic group. (C) The graph demonstrates the levels of collagen-I (arbitrary units) per group. CB: 0.46 ± 0.05, Ex-4: 0.26 ± 0.03, D: 0.48 ± 0.07, D+Ex-4: 0.89 ± 0.08, ##p < 0.01 versus diabetic group. (D) The graph demonstrates the levels of fibronectin (arbitrary
21
units) per group. CB: 0.33 ± 0.07, Ex-4: 0.32 ± 0.02, D: 0.69 ± 0.01, D+Ex-4: 0.36 ± 0.05, *p < 0.05 versus control group and #p < 0.05 versus diabetic group. (E) The graph demonstrates the levels of TGF-β1 (arbitrary units) per group. CB: 0.84 ± 0.04, Ex-4: 0.73 ± 0.04, D: 0.62 ± 0.16, D+Ex-4: 0.41 ± 0.01, *p < 0.05 and **p < 0.01 versus control group; Ψp < 0.05 versus Ex-4-injected group; and #p < 0.05 versus diabetic group. Values are mean ± S.E.M. n=5 animals for each group. CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4:
A
CC
EP
TE
D
M
A
N
U
SC RI PT
Exendin-4-injected diabetic group.
Fig. 7. (A) Western immunoblot of p-IRS1(Ser307), SOCS3 and p-AKT (Ser473) in lung homogenates. (B) The graph demonstrates the levels of p-IRS1(Ser307) (arbitrary units) per group. CB: 1.06 ± 0.06, Ex-4: 0.55 ± 0.04, D: 1.2 ± 0.08, D+Ex-4: 1.79 ± 0.1, *p < 0.05 versus control group; and #p < 0.05 versus diabetic group. (C) The graph demonstrates the levels of SOCS3 (arbitrary units) per group. CB: 0.92 ± 0.05, Ex-4: 0.94 ± 0.08, D: 1.55 ± 0.07,
22
D+Ex-4: 0.63 ± 0.05, ***p < 0.001 versus control group; and ###p < 0.001 versus diabetic group. (D) The graph demonstrates the levels of p-AKT (Ser473) (arbitrary units) per group. CB: 0.65 ± 0.07, Ex-4: 0.51 ± 0.08, D: 0.84 ± 0.14, D+Ex-4: 0.62 ± 0.11, #p < 0.05 versus diabetic group. Values are mean ± S.E.M. n=5 animals for each group. CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-injected diabetic
A
CC
EP
TE
D
M
A
N
U
SC RI PT
group.
23
Table 1. List of antibodies used for immunocytochemistry and Western blotting. Dilution
Application
Company
Catalog number
PCNA
1:50
Immunohistochemistry
Thermo Scientific
RB-9055-P
Cleaved Caspase-3
1:100
Westen blotting
Millipore
AB3623
1:100
Immunohistochemistry
CST
Pro-SPC
1:5000
Western blotting
Millipore
AB3786
RAGE
1:500
Western blotting
Novus Biologicals
NBP-1-42612
α-SMA
1:500
Western blotting
Santa Cruz
sc-32251
Collagen
1:1000
Western blotting
Novus Biologicals
NBP-1-30054
Fibronectin
1:10000
Western blotting
BD Biosciences
BD-610077
TGF-β
1:500
Western blotting
p-IRS1
1:1000
Western blotting
SOCS3
1:1000
p-AKT
A
N
U
SC RI PT
Antibody
9661S
sc-146
CST
2381S
Western blotting
CST
2932S
1:1000
Western blotting
CST
4060-S
β-actin
1:500
Western blotting
Santa Cruz
sc-47778
Goat antimouse IgG
1:5000
Western blotting
Abcam
ab98679
Western blotting
Santa Cruz
sc-2004
EP
TE
D
M
Santa Cruz
1:5000
A
CC
Goat anti-rabbit IgG
24
S0196-9781(17)30376-5 https://doi.org/10.1016/j.peptides.2017.12.007 PEP 69885
To appear in:
Peptides
Received date: Revised date: Accepted date:
17-7-2017 4-12-2017 6-12-2017
Please cite this article as: Oztay Fusun, Bas Serap Sancar, Gezginci-Oktayoglu Selda, Ercin Merve, Bolkent Sehnaz.Exendin-4 partly ameliorates of hyperglycemiamediated tissue damage in lungs of streptozotocin-induced diabetic mice.Peptides https://doi.org/10.1016/j.peptides.2017.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TITLE PAGE
Title. Exendin-4 partly ameliorates of hyperglycemia-mediated tissue damage in lungs of streptozotocin-induced diabetic
SC RI PT
mice
Author names and affiliations.
Fusun Oztay, Serap Sancar Bas, Selda Gezginci-Oktayoglu, Merve Ercin, Sehnaz Bolkent
U
Fusun Oztay, MSc, PhD, Prof.
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134,
A M
Serap Sancar Bas, MSc, PhD, Assistant Prof.
N
ISTANBUL/TURKEY, e -mail: [email protected]
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134,
TE
D
ISTANBUL/TURKEY, e -mail: [email protected]
EP
Selda Gezginci-Oktayoglu, MSc, PhD, Assoc. Prof. Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134,
CC
ISTANBUL/TURKEY, e -mail: [email protected]
A
Merve Ercin, MSc
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134, ISTANBUL/TURKEY, e -mail: [email protected]
Sehnaz Bolkent MSc, PhD, Prof.Dr.
1
Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134, ISTANBUL/TURKEY, e-mail: [email protected]
Corresponding author.
SC RI PT
Selda Gezginci-Oktayoglu, MSc, PhD, Assoc. Prof. Istanbul University, Faculty of Science, Biology Department, Molecular Biology Section, Vezneciler, 34134, ISTANBUL/TURKEY, e -mail: [email protected] Phone:+90 212 455 57 00 (external, 15084)
U
Fax: +90 212 528 05 27
A
CC
EP
TE
D
M
A
N
Graphical abstract
2
Highlights
Exendin-4 contributed to the healing of lung against hyperglycemiamediated increased oxidative stress, tissue damage and pulmonary edema through its glucose-lowering and oxidative stress-reducing effects.
SC RI PT
Exendin-4 induced the cell proliferation in pulmonary epithelium and interstitium.
Exendin-4 results in the disruption of insulin signaling and increases at the protein level of collagen-type-I in the lung of diabetic mice.
N
U
Exendin-4-mediated a critical reduction in the amount of fibronectin may result in excessive collagen accumulation in the lung of diabetic mice.
A
ABSTRACT
M
Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion, and plays anti-inflammatory role in atherosclerosis, and has surfactant-releasing effects in lungs. GLP-1 analogues are used in diabetes therapy. This is the first study to investigate the effects of exendin-4, a GLP-1 receptor agonist, on lung injury in diabetic mice. BALB/c male mice
D
were divided into four groups. The first group was given only citrate buffer, the second group was given only
TE
exendin-4, the third group was given only streptozotocin (STZ), and the fourth group was given both exendin-4 and STZ. Exendin-4 (3 µg/kg) was administered daily by subcutaneous injection for 30 days after mice were rendered diabetic with a single dose of STZ (200 mg/kg). Structural alterations, oxidative stress, apoptosis, insulin signaling
EP
and expressions of prosurfactant-C, alpha-smooth muscle actin, collagen-I and fibronectin were evaluated in lung tissue. Diabetic mice lungs were characterized by induced oxidative stress, apoptosis, edema, and cell proliferation.
CC
They had honeycomb-like alveoli, thick alveolar walls, and hypertrophic pneumocytes. Although exendin-4 treatment improved pulmonary edema, apoptosis, oxidative stress, and lung injury, it led to the disrupted insulin signaling and interstitial collagen accumulation in the lungs of diabetic mice. Exendin-4 reduced hyperglycemia-
A
mediated lung damage by reducing glucose, and oxidative stress and stimulating cell proliferation. However, exendin-4 led to increased risk by reducing insulin signaling in the lungs and collagen accumulation around pulmonary vasculature in diabetic mice.
Keywords: Exendin-4, diabetes, lung injury, insulin resistance, fibrosis.
3
1. Introduction
Type 2 diabetes, obesity and metabolic syndrome are common comorbidities of some chronic lung diseases [40].
SC RI PT
Clinical and experimental studies on diabetic animals and patients have reported a reduction in lung elastic recoil, lung-diffusing capacity, and pulmonary capillary blood volume, as well as induced pulmonary oxidative stress,
structural damage of alveolar areas, and thicker alveolar basement membranes [33, 35]. The disruption of insulin signaling is a common feature of the metabolic diseases mentioned above and it is considered to be a strong risk
factor for lung diseases [37]. Type 2 diabetes is a metabolic disease characterized by insulin resistance, which arises from impaired insulin signaling in insulin-sensitive cells. Insulin signaling is triggered by the insulin receptor, which is a kinase that phosphorylates the tyrosine residues of insulin receptor substrate (IRS) that in turn activates AKT
U
with the phosphorylation of its serine residues. Insulin resistance develops as a result of long-term high-level insulin levels in the blood and is characterized primarily by an increase in phospho (p)-IRS1 (ser307) and a decrease in p-
N
AKT (ser473) in cells [5]. Although the lack of cytokine signaling-3 (SOCS3) is generally considered to play a negative
A
role in this mechanism [5], there is also evidence that insulin resistance progresses in the absence of this molecule [41]. The presence of insulin receptors in the lungs was demonstrated years ago [43]. The negative effects of
M
gestational diabetes on fetal lung development are related to increased maternal insulin levels [2]. In addition, disrupted insulin signaling has been reported to negatively affect lung function in adolescents [17]. Moreover, high
D
levels of insulin have been reported to induce contraction in the airway smooth muscle [34], and insulin increases the extracellular matrix proteins by stimulating the proliferation and contraction of the airway smooth muscle [9].
TE
Hence, diabetes may contribute to pulmonary complications but, studies on the molecular mechanism of lung
EP
dysfunction in diabetics are insufficient.
Glucagon-like peptide-1 (GLP-1) stimulates mainly glucose-induced insulin secretion and reduces glucagon
CC
release [23, 30]. GLP-1 and its analogues can increase beta-cell mass in animal models with type 2 diabetes and human islet cells [19, 42]. Exendin-4, a GLP-1 receptor agonist, regulates insulin production and glycemic control in
A
diabetes [20]. Exendin increases insulin secretion [10]. For these reasons, exendin-4 has been used to treat type 2 diabetic patients. GLP-1 releases surfactant in the lungs [44]. GLP-1 receptors have reduced mortality and improved lung function in a model of experimental obstructive lung disease in female mice [45]. However, the therapeutic effect of GLP-1 and its receptor agonists on diabetes-mediated lung damage is unclear. The present study examines the therapeutic effect of exendin-4, which has similar functional properties to native GLP-1, on lung injury in diabetic mice. The study evaluates lung tissue for the following: structural alterations, oxidative stress, apoptosis, and insulin signaling as well as expressions of prosurfactant-C (pro-SPC, type II pneumocyte marker), alpha-smooth
4
muscle actin (α-SMA, myofibroblast marker) collagen-I, fibronectin, and insulin signaling regulators (p-IRS1, p-AKT and SOCS3).
2. Materials and methods
SC RI PT
2.1. Animals and experimental design All experiments were performed in accordance to the guidelines of the Istanbul University Local Ethic Committee of Experimental Animals. The mice were provided from Experimental Medical Research Institute of Istanbul
University. They were kept at a stable temperature (22 ± 1ºC) for 12 h light and dark cycles and fed with a standard pellet chow.
BALB/c male mice at 10 weeks of age were divided into four groups (10 animals per group). The first group was given citrate buffer only, the second group was administered exendin-4 alone, the third group received
U
streptozotocin (STZ), and the fourth group was given both STZ and exendin-4. The exendin-4 (Sigma, St. Louis, MO,
N
3 μg/kg dissolved in ultrapure water) was daily administered by subcutaneous injection for 30 days after mice were rendered diabetic by administration of a single dose of STZ (Sigma, St. Louis, MO, 200 mg/kg dissolved in 0.01 M
A
citrate buffer, pH: 4.5). Vehicle or exendin-4 was injected to control animals once every 24 hours. The animals were
M
fasted overnight prior to the experiments, but they were allowed to drink water. On day 30, the animals were
TE
2.2. Histopathological evaluation
D
sacrificed by cervical dislocation and the lung tissues were taken immediately.
Pieces of lung tissue were fixed in Bouin’s solution. The tissues were washed with 70% ethanol to remove picric acid, dehydrated in graded ethanol series and embedded in paraffin wax. Section of 4 µm thickness were taken
EP
serially and stained with Masson’s trichrome. Microscopic analysis of tissues was made using a light microscope (Olympus, CX41) and the photomicrographs were taken with Olympus DP71 digital camera. The lung injury was
CC
evaluated in the lung sections according to the following criteria: disintegrity of pulmonary epithelium, structural alterations in alveolar areas and bronchioles, the accumulation of collagen in the connective tissue, and the
A
presence of the pulmonary inflammation and edema.
2.3. Immunohistochemistry Sections of lungs, mounted on poly-L-lysine slides, were cleared in toluene, hydrated through decreasing ethyl alcohol series and boiled in 10 mM citrate buffer (pH 6), [27]. After incubation in 3% H2O2 in methanol and 0.3 %Triton-X 100, sections were incubated with anti-proliferative cell nuclear antigen (PCNA) antibody for 30 min at
5
room temperature or anti-cleaved caspase-3 antibody at 4°C overnight. The antibodies used are listed in Table 1. The immunostaining was carried out by using the Histostain Plus Kit (Invitrogen, Paisley, Scotland) comprising the streptavidin-biotin-horseradish peroxidase method, and revealed using 3-amino-9-ethylcarbazole as chromogen. Percentage of stained cells was quantified by following formula: (Ʃ immunopositive cell / Ʃ cell) x 100.
SC RI PT
2.4. Western blotting
The lungs were homogenized in cold RIPA buffer. The homogenates were centrifuged for 10 minutes at 4°C and 10.000g. The supernatant was removed for electrophoresis. Equal amounts of protein (80 μg) were separated on SDS-PAGE gels (7.5%, 10% and 12%) and transferred to nitrocellulose membrane. The blots were blocked for nonspecific binding for 1 hour at room temperature in 5% fat-free dry milk in TBST. Thereafter the blots were
incubated at 4°C with primary antibodies overnight. The antibodies are listed in Table 1. After washing, specific
U
bands were visualized with the respective horseradish peroxidase-conjugated secondary antibodies (Table1) and
M
2.5. Measurement of oxidative stress
A
Imaging Software and normalized to β-actin protein bands.
N
Luminol reagent (Santa Cruz, CA, USA). The intensities of protein bands were quantified using the Kodak Molecular
Tissue reactive oxygen species (ROS) generation was measured according to the method of Lebel and Bondy
D
[24] further modified by Kim et al [22]. ROS production was measured by the oxidation of a poorly fluorescent non-
TE
ionic, non-polar reagent 2',7'-dichlorofluorescein diacetate (DCFH-DA) into the highly fluorescent 2',7'dichlorofluorescein. Briefly, lung tissue homogenates containing 50 µg protein were transferred to black 96-well microplates and taken to 100 μl with assay medium (20 mM Tris -HCl, 130 mM KCl, 5 mM MgCl2, 20 mM NaH2PO4,
EP
30 mM glucose). DCFH-DA (10 µg/ml, 10 µl volume) dissolved in dimethylsulfoxide, was added to the mixture in the dark. After incubation at 37ºC for 3h, fluorescent product was measured at 485 nm excitation and 525 nm emission
CC
wavelength by a spectrofluorometer. Then, ROS generation was calculated as the % of control. In addition, oxidative damage, was determinated by measuring malondialdehyde (MDA) levels according to the protocol described by Ledwozwy and coworkers [25].
A
2.6. Statistical analysis Data are expressed as mean ± S.E.M. The results were analyzed by one-way ANOVA followed by Tukey’s post
hoc test to compare differences among groups by using GraphPad Prism software, version 5.00 (San Diego, CA).
3. Results
6
3.1. Exendin-4 treatment reduced lung injury in diabetic mice Fasting blood glucose values were measured with a glucometer on day 0 and day 30 of the experiment, and individuals with values above 126 mg/dL were considered diabetic according to a previous report [6]. The fasting blood glucose concentration was 99.1 ± 2.3 mg/dL in control mice, 92.3 ± 1.6 mg/dL in exendin-4 treated mice,
SC RI PT
380.7 ± 15.7 mg/dL in STZ-injected mice, and 115.1 ± 3.19 mg/dL in exendin-4 injected diabetic mice at day 30 [20]. These results indicate that administration of STZ causes the development of advanced diabetes at the indicated dose and time.
Diabetes induced the disruption of alveolar structure. Also, diabetic mice had honeycomb-like alveoli, thick alveolar walls and hypertrophic pneumocytes in their lungs. It was detected fluid accumulation in the tissue.
U
Exendin-4 treatments resulted in the regression of edema and healing of alveolar structure. However, increase in
N
cell number around bronchioles and vessels, and interstitial collagen accumulation were evident in diabetic mice
M
A
treated with exendin-4 (Fig. 1).
3.2. Exendin-4 treatment reduced oxidative stress seen in the diabetic lungs.
D
The total ROS levels in the lung tissues from diabetic animals were higher than the control group (p < 0.05), and treatment of diabetic mice with exendin-4 normalized ROS levels (p < 0.05) (Fig. 2A). On the other hand,
TE
exendin-4 and STZ-induced diabetes caused an increase in the levels of MDA versus control group (p < 0.05, p < 0.001 respectively). Treatment of diabetic mice with Exendin-4 ameliorated MDA levels only in the Exendin-4
EP
injected group (p < 0.01) (Fig. 2B).
CC
3.3. Exendin-4 treatment reduced apoptotic cells in the diabetic lungs The levels of active caspase-3 were higher in lung tissue in diabetic group versus the control group (p < 0.001)
and exendin-4 reduced the levels of this active enzyme versus diabetic group (p < 0.01) (Figs. 3A and B). In
A
agreement with these results, the apoptotic cells were identified as pulmonary epithelial and interstitial cells in alveolar areas of diabetic mice under light microscope and the number of these cells was reduced with exendin-4 treatment (Fig. 3C). 3.4. Exendin-4 treatment increased the levels of pro-SPC and RAGE proteins in the diabetic lungs Diabetes did not significant alter the levels of pro-SPC and receptor of advanced glycation end-products proteins (RAGE, a marker of type I pneumocyte) in the lung (p > 0.05) (Figs. 4A and B). However, exendin-4
7
treatment increased the levels of these proteins in diabetic mice (p < 0.001 and p < 0.01, respectively) (Figs. 4A and B).
3.5. Exendin-4 increased cell proliferation in the diabetic lungs
SC RI PT
There was not any significant changes in the number of PCNA-immunoreactive (PCNA+) cells in diabetic animals versus control group (p > 0.05). However, exendin-4 treatment to diabetic group caused an increase in PCNA+ cell number versus diabetic mice (p < 0.05), exendin-4 injected control mice (p < 0.05) and vehicle-injected control mice (p < 0.01). Increase in PCNA+ cell number around bronchioles and venules was observed in diabetic mice treated with exendin-4 (Figs. 5A and B).
U
3.6. Exendin-4 induced the production and accumulation of collagen-I in the diabetic lungs
N
Diabetic mice were characterized by the decreased α-SMA (p < 0.01) and active TGFβ-1 (p < 0.05) levels and the increased fibronectin levels (p < 0.05) versus control group (Figs. 6A-E). The significant increase in the levels of
A
collagen-I protein could not be determined in the lungs of diabetic mice (Figs. 6A-E). Exendin-4 increased the levels
M
of collagen-I proteins (p <0.01) and α-SMA proteins in the lung of diabetic mice (Figs. 6A-C). Exendin-4-mediated collagen-I production was confirmed by microscopic finding such as interstitial collagen accumulation around pulmonary vessels. However, fibronectin (p < 0.05) and active TGFβ-1 (p < 0.05) levels were decreased by exendin-4
TE
D
treatments in the lung tissue of diabetic mice (Figs. 6D and E).
EP
3.7. Exendin-4 reduced insulin signaling in the diabetic lungs We did not observe the disrupted insulin signaling, which can occur in absence of alteration in p-IRS1 (p > 0.05), SOCS3 (p < 0.001) and p-AKT levels in the lung of diabetic group in comparison with the control group.
CC
However, exendin-4 treatment in diabetic mice caused an increase in p-IRS1 (p < 0.05), decrease in SOCS3 (p<0.001) and p-AKT levels (p < 0.05) in comparison with the diabetic group, indicating the disruption of insulin signaling. In contrast, exendin-4 treatment in control mice resulted in a decrease in p-IRS1 level (p < 0.05)
A
compared to the control group, which suggested that exendin-4 may reduce insulin signaling in lung only under hyperglycemic condition (Figs. 7A-C).
4. Discussion
8
Diabetes-induced lung damage is generally disregarded by patients and physicians, because it is subclinical. However, studies on the lungs of diabetic animals have shown enlargement of air sacs, alveolar wall thickening, structural damage of alveoli, intense inflammatory reactions, and increased pulmonary edema as well as reductions in surfactant proteins and lung-diffusing capacity [1, 13, 28, 33]. Diabetic lungs are characterized by the overproduction of ROS, reactive nitrogen species, secondary ends of lipid peroxidation such as MDA and a decrease
SC RI PT
in the capacity of the pulmonary antioxidant defense system and oxidative stress [16, 26]. Xiong et al. [48] reported elevated MDA levels in serum of diabetic mice. In the lungs they noted hyperglycemia-induced inflammatory
mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 and interleukin-10. An increase in oxidants and a decreased in pulmonary antioxidant enzymes lead to increased cellular stress and consequently, structural damage develops in diabetic lungs. In the present study, hyperglycemia led to similar structural damage and alterations of oxidative stress parameters in diabetic mice lungs. Our study and others, suggest that increased ROS and MDA
levels, and induced structural lung damage are hallmarks of hyperglycemia-mediated alterations in diabetic mice
U
lungs. It is well known that exendin-4 regulates insulin production and glycemic control in diabetes [10, 20].
Gezginci-Oktayoglu and Bolkent [20] showed that exendin-4 increased β-cell counts and insulin production in mice.
N
Exendin-4 administered to diabetic mice significantly reduced blood glucose, the levels of interleukin-1β and TNF-α,
A
and oxidative stress in kidneys [36]. Similarly, in the present study focused on diabetic mice lungs, regression of hyperglycemia via exendin-4 administration attenuated hyperglycemia-mediated abnormal structural alterations
D
M
and oxidative stress.
Chronically high glucose uptake can lead to glucotoxicity and cell death. When high glucose is consumed by
TE
the mitochondrial electron transport system, ROS production increases. Proteins released from mitochondria into the cytosol and the subsequent caspase-3 activation promote cell apoptosis [38]. In diabetic rat lungs, high glucose induces apoptosis through redox imbalance, mitochondrial abnormalities, and caspase-3 activation [47]. These data
EP
are confirmed by several findings in the present study such as the presence of many cleaved-caspase-3+ cells in the pulmonary epithelium and interstitium and increases in cleaved-caspase-3 protein, ROS, and MDA in lung tissue.
CC
Thus, the present study supplies valuable data on hyperglycemia-mediated damage to the lungs. The pulmonary epithelium covering the inner surface of the alveoli is very sensitive to oxidative stress. It consists of two types of epithelial cells, type I and II pneumocytes. Type II pneumocytes, which produce surfactant proteins, play a role in
A
regenerating pulmonary epithelium by supporting its survival after injury. They proliferate and subsequently differentiate into pulmonary epithelium cells as a response to tissue damage [14]. In agreement with this the present study shows pulmonary epithelial cell hyperplasia, increased cleaved-caspase-3+ and PCNA+ cells in the pulmonary epithelium and interstitium which are likely signs of diabetes-mediated tissue damage. We postulate that diabetes induces cell apoptosis and proliferation in both pulmonary epithelium and interstitium in mice lungs as a response to tissue damage. On the other hand, the pulmonary epithelium is close to pulmonary capillaries. Cell
9
junctions between pulmonary epithelial cells prevent accumulation of fluids in alveolar areas. Diabetic complications in lung tissue are related to pulmonary epithelial and endothelial dysfunctions. Diabetes-induced hyperglycemia and increased oxidative stress cause epithelial cell death and vascular permeability [7]. The present study clearly demonstrates pulmonary edema in diabetic mice lungs. Based on previous studies and the present one we suggest that diabetes-induced pulmonary epithelial damage as well as pulmonary epithelial and endothelial
SC RI PT
dysfunction lead to pulmonary edema. Exendin-4 in diabetic mice ameliorated of hyperglycemia-mediated increased oxidative stress, tissue damage and pulmonary edema in lung tissue because it reduces glucose and
oxidative stress. Diabetic mice who received exendin-4 had fewer cleaved-caspase-3+ cells, reduced levels of ROS and MDA, more PCNA+ cells, and RAGE and pro-SPC proteins. RAGE and pro-SPC proteins are cell markers of type I and II pneumocytes, respectively. Hence, the increases in RAGE and pro-SPC proteins interpreted as repair of the
pulmonary epithelium. Thus, we suggest that exendin-4 contributes to the repair of the pulmonary epithelium and interstitium by stimulating cell proliferation. A proliferative effect of exendin-4 was also reported by Baggio and
U
Drucker [3]. They noted that exendin-4 induced pancreatic β-cell proliferation in rodents and ameliorated glucose
A
preosteoblasts via activation of the MAPK pathway [15].
N
metabolism in diabetic animals and patients. It was also shown that exendin-4 promoted cell proliferation of
M
In diabetes extracellular matrix components accumulate and the basement membrane thickens in the lungs and kidneys [18, 46]. As a complication of diabetes, fibrosis can occur in these tissues as well as diabetic
D
nephropathy and retinopathy [4]. Tissue fibrosis is characterized by an increase fibroblasts and myofibroblasts expressing α-SMA and an accumulation of extracellular matrix components such as collagens, fibronectin,
TE
proteoglycans, and hyaluronic acid in the matrix [32, 49]. This accumulation and excessive production are modulated by several growth factors and TGF-β [8]. Enomoto et al. [12] reported that diabetes may be a risk factor for idiopathic pulmonary fibrosis. However, instead of a fibrotic response, thickening basement membranes
EP
were observed in diabetic lungs. Zheng et al. [50] detected basement membrane thickening of the alveolar wall through excessive production and accumulation of collagen-IV. In the present study, diabetic mice that did not
CC
receive exendin-4 did not exhibit fibrotic alterations in the lungs. They did not have increased levels of activatedTGF-β (a profibrotic cytokine), α-SMA and collagen-I proteins, except for fibronectin. In contrast, mice that did receive exendin-4 showed greater production and accumulation of collagen-I protein around the pulmonary
A
vasculature. These mice showed lower levels of activated-TGFβ and fibronectin, which is present in these areas. Although TGF-β is an important profibrotic cytokine, the production and accumulation of extracellular matrix components can be stimulated independently of TGF-β [31]. Kawelke et al. [21] reported that collagen-I production was more pronounced by fibronectin-deficient hepatic stellate cells. They found that removing fibronectin from the liver and hepatic stellate cells led to an increase in baseline collagen-I as a response to induced experimental fibrosis. On the basis of these data, we postulate that fibronectin regulates collagen production. In
10
our study, exendin-4 mediated collagen production and accumulation around the pulmonary vasculature can be related to the reduction of fibronectin in diabetic mice lungs. Furthermore, exendin-4 induced the production and accumulation of collagen by stimulating mesenchymal cell proliferation in diabetic mice. This was confirmed by the presence of PCNA+ cells in areas where interstitial collagen is deposited around the pulmonary vasculature in the
SC RI PT
diabetic mice who received exendin-4.
Lung epithelial cells strongly express insulin receptor in the pseudoglandular stage. In contrast, insulin
receptors decrease in more advanced stages of development [39]. Disruption of insulin signaling has been reported to negatively affect lung function of adolescents [Forno et al., 2015]. This distribution can be tracked with SOCS3, pIRS1, and p-AKT proteins [29]. Insulin leads to the activation of IRS proteins when bound to their receptors, then
these proteins direct many cellular events namely growth and survival in different cell types via PI3K/AKT signaling [Singh et al., 2013]. An increase in serine 307 phosphorylation of IRS1 is the sign of disrupted insulin signaling [5], in
U
contrast, tyrosine phosphorylation of IRS1 leads to insulin signaling. SOCS3 is a regulator of insulin signaling
N
especially induced by cytokines and insulin [11]. In the present study, exendin-4 treatment caused an increase in IRS1 phosphorylation and a decrease in SOCS3, which means that the disruption of insulin signaling occurred in the
A
lungs. Although exendin-4 administration to diabetic mice contributed to lungs healing of tissue damaged by
M
hyperglycemia, the disruption of insulin signaling may have a negative impact on lung biology.
D
6. Conclusion
TE
Diabetes has major impacts on the lungs, where it induces oxidative stress, apoptotic cell death, edema, and disrupts the alveolar structure. Exendin-4 administration to diabetic mice contributes to the healing of lung against hyperglycemia-mediated increased oxidative stress, tissue damage and pulmonary edema by its glucose-lowering
EP
and anti-oxidant effects. Additionally, it induced tissue repair in pulmonary epithelium and interstitium by stimulating cell proliferation. Nevertheless, exendin-4 disrupted insulin signaling and increased protein levels of
CC
collagen-I in the lungs of diabetic mice. Unexpectedly, it decreased fibronectin protein levels. Exendin-4-mediates a critical reduction in the amount of fibronectin that may result in excessive collagen accumulation around the
A
pulmonary vasculature.
Conflict of interest The authors declare that they have no conflict of interest to disclose.
11
Acknowledgements This work was supported by Scientific Research Projects Coordination Unit of Istanbul University with the project number 408. The authors thank to Dr. Anna Cosagliola from Napoli Federico II University for editing of the
SC RI PT
manuscript.
References
[1] Alkan M, Celik A, Bilge M, Kiraz HA, Kip G, Ozer A, et al. The effect of levosimendan on lung damage
after myocardial ischemia reperfusion in rats in which experimental diabetes was induced. J Surg Res 2014;
U
193:920-25.
N
[2] Azad MB, Becker AB, Kozyrskyj AL. Association of maternal diabetes and child asthma. Pediatr Pulmonol
A
2013; 48:545-552.
M
[3] Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007; 132:2131–57.
D
[4] Ban CR, Twigg SM. Fibrosis in diabetes complications: pathogenic mechanisms and circulating and urinary
TE
markers. Vasc Health Risk Manag 2008; 4:575-96.
[5] Boucher J, Kleinridders A, Kahn CR. Insulin receptor signaling in normal and insulin-resistant states. Cold
EP
Spring Harb Perspect Biol 2014; 6(1). pii: a009191. doi: 10.1101/cshperspect. a009191.
CC
[6] Cefula W. Standards of medical care in diabetes-2014: Summary of revisions. Diabetes Care 2014; 38:1-93.
[7] Clemmer JS, Xiang L, Lu S, Mittwede PN, Hester RL. Hyperglycemia-mediated oxidative stress increases
A
pulmonary vascular permeability. Microcirculation 2016; 23:221-9.
[8] Cutroneo KR, White SL, Phan SH, Ehrlich HP. Therapies for bleomycin induced lung fibrosis through regulation of TGF-beta1 induced collagen gene expression. J Cell Physiol 2007; 211:585-9.
[9] Dekkers BG, Schaafsma D, Tran T, Zaagsma J, Meurs H. Insulin-induced laminin expression promotes a hypercontractile airway smooth muscle phenotype. Am J Respir Cell Mol Biol 2009; 41:494-504.
12
[10] Egido EM, Silvestre RA, Hernández R, Marco J. Exendin-4 dose-dependently stimulates somatostatin and insulin secretion in perfused rat pancreas. Horm Metab Res 2004; 36(9):595-600.
[11] Emanuelli B, Peraldi P, Filloux C, Sawka-Verhelle D, Hilton D, Van Obberghen E. SOCS-3 is an insulininduced negative regulator of insulin signaling. J Biol Chem 2000; 275:15985-91.
SC RI PT
[12] Enomoto T, Usuki J, Azuma A, Nakagawa T, Kudoh S. Diabetes mellitus may increase risk for idiopathic pulmonary fibrosis. Chest 2003; 123(6):2007-11.
[13] Eren G, Cukurova Z, Hergunsel O, Demir G, Kucur M, · Uslu E, et al. Protective effect of the nuclear factor kappa B inhibitor pyrrolidine dithiocarbamate in lung injury in rats with streptozotocin-induced diabetes. Respiration 2010; 79: 402–10.
U
[14] Evans MJ, Cabral LJ, Stephens RJ, Freeman G. Renewal of alveolar epithelium in the rat following
N
exposure to NO2. Am J Pathol 1973; 70(2):175-98.
A
[15] Feng Y, Su L, Zhong X, Guohong W, Xiao H, Li Y, Xiu L. Exendin-4 promotes proliferation and
M
differentiation of MC3T3-E1 osteoblasts by MAPKs activation. J Mol Endocrinol 2016; 56:189-99.
[16] Forgiarini LA. Jr Kretzmann NA, Porawski M, Dias AS, Marroni NA. Experimental diabetes mellitus:
D
oxidative stress and changes in lung structure. J Bras Pneumol 2009; 35:788-91.
TE
[17] Forno E, Han YY, Muzumdar RH, Celedón JC. Insulin resistance, metabolic syndrome, and lung function in US adolescents with and without asthma. J Allergy Clin Immunol 2015; 136:304-11.
EP
[18] Friedman E. Diabetic renal disease. In: Rifkin H, Porte D Jr, eds. Ellenberg and Rifkin's Diabetes Mellitus:
CC
Theory and Practice. 4th ed. New York, NY: Elsevier; 1990;684–709.
[19] Garber AJ. Incretin effects on β-cell function, replication, and mass: The human perspective. Diabetes Care
A
2011; 34:258–63.
[20] Gezginci-Oktayoglu S, Bolkent S. Exendin-4 exerts its effects through the NGF/p75NTR system in diabetic mouse pancreas. Biochem Cell Biol 2009; 87:641–51.
[21] Kawelke N, Vasel M, Sens C, Au Av, Dooley S, Nakchbandi IA. Fibronectin protects from excessive liver fibrosis by modulating the availability of and responsiveness of stellate cells to active TGF-β. PLoS One 2011; 6(11):28181.
13
[22] Kim JD, McCarter RJ, Yu BP. Influence of age, exercise, and dietary restriction on oxidative stress in rats. Aging 1996; 8:123-29.
[23] Kolterman OG, Buse JB, Fineman MS, Gaines E, Heintz S, Bicsak TA, et al. Synthetic exendin-4 (AC2993) significantly reduces postprandial and fasting plasma glucose in subjects with type 2 diabetes. J
SC RI PT
Clin Endocrinol Metab 2003; 88:3082–89.
[24] Lebel CP, Bondy SC. Sensitive and rapid quantitation of oxygen reactive species formation in rat synaptosomes. Neurochem Int 1990; 17:435-40.
[25] Ledwozwy A, Michalak J, Stepien A, Kadziolka A. The relationship plasma triglycerides, cholesterol, total
U
lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986; 155: 275-83.
[26] Liang JQ, Ding CH, Ling YL, Xu HB, Lu P, Xian XH. The protective function of puerarin to the injury of
A
N
the lung and its mechanisms during diabetes. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2007; 23:355-8.
[27] Liguori G, Pavone LM, Assisi L, Langella E, Tafuri S, Mirabella N, et al. Expression of orexin B and its
M
receptor 2 in rat testis. Gen Comp Endocrinol 2017; 242:66-73.
TE
Intern Med 2006; 20:47-51.
D
[28] Mexas AM, Hess RS, Hawkins EC, Martin LD. Pulmonary lesions in cats with diabetes mellitus. J Vet
[29] Nicolau J, Lequerré T, Bacquet H, Vittecoq O. Rheumatoid arthritis, insulin resistance and diabetes. Joint
EP
Bone Spine 2016; 1297-319X(16)30153-1.
[30] Nielsen LL, Young AA, Parkes DG. Pharmacology of exenatide (synthetic exendin-4): a potential
CC
therapeutic for improved glycemic control of type 2 diabetes. Regul Pept 2004; 117:77-88.
[31] Oga T, Matsuoka T, Yao C, Nonomura K, Kitaoka S, Sakata D, et al. Prostaglandin F(2alpha) receptor
A
signaling facilitates bleomycin-induced pulmonary fibrosis independently of transforming growth factorbeta. Nat Med 2009; 15:1426-30.
[32] Ovet H, Oztay F. The copper chelator tetrathiomolybdate regressed bleomycin-induced pulmonary fibrosis in mice, by reducing lysyl oxidase expressions. Biol Trace Elem Res 2014; 162:189-99.
14
[33] Oztay F, Kandil A, Gurel E, Ustunova S, Kapucu A, Balci H, et al. Relation between nitric oxide and leptin in the lung of rat with streptozotocin-induced diabetes. Cell Biochem Funct 2007; 26:162–71.
[34] Papagianni M, Hatziefthimiou A, Chachami G, Gourgoulianis K, Molyvdas PA, Paraskeva E. Insulin causes a transient induction of proliferation via activation of the PI3-kinase pathway in airway smooth
SC RI PT
muscle cells. Exp Clin Endocrinol Diabetes 2007; 115:118-23.
[35] Ramirez LC, Dal Nogare A, Hsia C, Arauz C, Butt I, Strowig SM, et al. Relationship between diabetes control and pulmonary function in insulin-dependent diabetes mellitus. Am J Med 1991; 91:371–6.
[36] Sancar-Bas S, Gezginci-Oktayoglu S, Bolkent S. Exendin-4 attenuates renal tubular injury by decreasing oxidative stress and inflammation in streptozotocin-induced diabetic mice. Growth Factors 2015; 33:419-
U
29.
[37] Singh S, Prakash YS, Linneberg A, Agrawal A. Insulin and the lung: connecting asthma and metabolic
A
N
syndrome. J Allergy (Cairo) 2013; 627384.
M
[38] Smart EJ, Li XA. Hyperglycemia: cell death in a cave. Biochim Biophys Acta 2007; 1772:524-6.
[39] Sodoyez-Goffaux FR, Sodoyez JC, De Vos CJ. Insulin receptors in the fetal rat lung. A transient
D
characteristic of fetal cells? Pediatr Res 1981; 15:1303-7.
TE
[40] Song Y, Klevak A, Manson JE, Buring JE, Liu S. Asthma, chronic obstructive pulmonary disease, and type 2 diabetes in the Women’s Health Study. Diabetes Res Clin Pract 2010; 90: 365–71.
EP
[41] Torisu T, Sato N, Yoshiga D, Kobayashi T, Yoshioka T, Mori H, et al. The dual function of hepatic SOCS3
CC
in insulin resistance in vivo. Genes Cells 2007; 12(2):143-54.
[42] Tourrel C, Bailbe´ D, Lacorne M, Meile MJ, Kergoat M, Portha B. Persistent improvement of type 2 diabetes in the Goto– Kakizaki rat model by expansion of the h-cell mass during the prediabetic period with
A
glucagon-like peptide-1 or exendin-4. Diabetes 2002; 51:1443–52.
[43] Ulane RE, Graeber JE, Steinherz R. A comparison of insulin receptors in the developing fetal lung in normal and in streptozotocin-induced diabetic pregnancies. Pediatr Pulmonol 1985; 1:86-90.
[44] Vara E, Arias-Diaz J, Garcia C, Balibrea JL, Blazquez E. Glucagon-like peptide-1(7–36) amide stimulates surfactant secretion in human type II pneumocytes. Am J Respir Crit Care Med 2001; 163:840–6.
15
[45] Viby NE, Isidor MS, Buggeskov KB, Poulsen SS, Hansen JB, Kissow H. Glucagon-like peptide-1 (GLP-1) reduces mortality and improves lung function in a model of experimental obstructive lung disease in female mice. Endocrinology 2013; 154:4503-11.
[46] Watanabe K, Senju S, Toyoshima H, Yoshida M. Thickness of the basement membrane of bronchial
SC RI PT
epithelial cells in lung diseases as determined by transbronchial biopsy. Respir Med 1997; 91(7):406-10.
[47] Wu J, Jin Z, Yan LJ. Redox imbalance and mitochondrial abnormalities in the diabetic lung. Redox Biol 2016; 11:51-9.
[48] Xiong XG, Wang WT, Wand LR, Jin LD, Lin LN. Diabetes increases inflammation and lung injury
U
associated with protective ventilation strategy in mice. Int Immunopharmacol 2012; 13:280–3.
[49] Yilmaz O, Oztay F, Kayalar O. Dasatinib attenuated bleomycin-induced pulmonary fibrosis in mice.
A
N
Growth Factors 2015; 33:366-75.
[50] Zheng F, Lu W, Wu F, Li H, Hu X, Zhang F. Recombinant decorin ameliorates the pulmonary structure
M
alterations by down-regulating transforming growth factor-beta1/SMADS signaling in the diabetic rats.
Figure Legends
TE
D
Endocr Res 2010; 35:35-49.
EP
Fig. 1. Lung histology of mice in the sections stained with Masson’s trichrome. In Exendin-4-treated diabetic mice, collagen fibers accumulated in the extracellular matrix seem to be colored green in the lung sections stained with
CC
Masson’s trichrome. Alveoli (A), terminal bronchiole (TB) and vein (V) honeycomb-like alveoli (□), edema (*) and thick alveolar walls (arrows). CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: diabetic group, and
A
D+Ex-4: Exendin-4-injected diabetic group.
16
SC RI PT U
N
Fig. 2. (A) The graph demonstrates the levels of ROS production (% of CB) per group. CB: 97.12 ± 16.5, Ex-4: 121.1 ±
A
6.95, D: 200.4 ± 20.17, D+Ex-4: 103.0 ± 32.97, *p < 0.05 versus control group; and #p < 0.01 versus diabetic group. (B) The graph demonstrates the levels of MDA (nmol/mg protein) per group. CB: 0.17 ± 0.04, Ex-4: 0.47 ± 0.09, D:
M
0.90 ± 0.06, D+Ex-4: 0.50 ± 0.03, *p < 0.05 versus control group; ***p < 0.001 versus control group, ## p < 0.01 versus diabe c group, ††p < 0.01 versus control group. Values are mean ± S.E.M. n=5 animals for each group. CB:
D
Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-injected diabetic
A
CC
EP
TE
group.
17
SC RI PT U
N
Fig. 3. (A) Western immunoblot of cleaved caspase-3 in lung homogenates. (B) The graph demonstrates the levels
A
of cleaved caspase-3 (arbitrary units) per group. CB: 0.22 ± 0.04, Ex-4: 0.26 ± 0.08, D: 0.82 ± 0.03, D+Ex-4: 0.44 ± 0.11, ***p < 0.001 versus control group; and ##p < 0.01 versus diabetic group. Values are mean ± S.E.M. n=5 animals
M
for each group. (C) Immunopositive reactions of active caspase-3 were shown by arrows for per group. CB: Vehicle-
A
CC
EP
TE
D
injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-injected diabetic group.
18
SC RI PT U N A M D TE EP CC A
Fig. 4. (A) Western immunoblot of pro-SPC and RAGE in lung homogenates. (B) The graph demonstrates the levels of pro-SPC (arbitrary units) per group. CB: 0.67 ± 0.05, Ex-4: 0.86 ± 0.02, D: 0.62 ± 0.01, D+Ex-4: 1.11 ± 0.01, *p < 0.05 and ***p < 0.001 versus control group; ΨΨp < 0.01 versus Ex-4-injected group; and ###p < 0.001 versus diabetic group. (C) The graph demonstrates the levels of RAGE (arbitrary units) per group. CB: 1.01 ± 0.01, Ex-4: 0.96 ± 0.03, D: 1.03 ± 0.01, D+Ex-4: 1.25 ± 0.0, ##p < 0.01 versus diabetic group. Values are mean ± S.E.M. n=5 animals for each
19
group. CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-
A
CC
EP
TE
D
M
A
N
U
SC RI PT
injected diabetic group.
Fig. 5. (A) PCNA+ lung cells (arrows) generally located around the veins (V). (B) The graph demonstrates the percent of PCNA+ lung cells per group. CB: 3.64 ± 0.83, Ex-4: 7.56 ± 1.3, D: 6.75 ± 1.6, D+Ex-4: 13.06 ± 0.62, #p < 0.05 versus diabetic group; **p < 0.01 versus control group. Values are mean ± S.E.M. n=5 animals for each group. CB: Vehicleinjected group, Ex-4: Exendin-4-injected group; D: Diabetic group, and D+Ex-4: Exendin-4-injected diabetic group.
20
SC RI PT U N A M D TE EP CC A
Fig. 6. (A) Western immunoblot of fibrosis markers that α-SMA, collagen-I and fibronectin in lung homogenates. (B) The graph demonstrates the levels of α-SMA (arbitrary units) per group. CB: 1.56 ± 0.05, Ex-4: 1.65 ± 0.11, D: 0.75 ± 0.09, D+Ex-4: 1.24 ± 0.02, **p < 0.01 versus control group; and #p < 0.05 versus diabetic group. (C) The graph demonstrates the levels of collagen-I (arbitrary units) per group. CB: 0.46 ± 0.05, Ex-4: 0.26 ± 0.03, D: 0.48 ± 0.07, D+Ex-4: 0.89 ± 0.08, ##p < 0.01 versus diabetic group. (D) The graph demonstrates the levels of fibronectin (arbitrary
21
units) per group. CB: 0.33 ± 0.07, Ex-4: 0.32 ± 0.02, D: 0.69 ± 0.01, D+Ex-4: 0.36 ± 0.05, *p < 0.05 versus control group and #p < 0.05 versus diabetic group. (E) The graph demonstrates the levels of TGF-β1 (arbitrary units) per group. CB: 0.84 ± 0.04, Ex-4: 0.73 ± 0.04, D: 0.62 ± 0.16, D+Ex-4: 0.41 ± 0.01, *p < 0.05 and **p < 0.01 versus control group; Ψp < 0.05 versus Ex-4-injected group; and #p < 0.05 versus diabetic group. Values are mean ± S.E.M. n=5 animals for each group. CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4:
A
CC
EP
TE
D
M
A
N
U
SC RI PT
Exendin-4-injected diabetic group.
Fig. 7. (A) Western immunoblot of p-IRS1(Ser307), SOCS3 and p-AKT (Ser473) in lung homogenates. (B) The graph demonstrates the levels of p-IRS1(Ser307) (arbitrary units) per group. CB: 1.06 ± 0.06, Ex-4: 0.55 ± 0.04, D: 1.2 ± 0.08, D+Ex-4: 1.79 ± 0.1, *p < 0.05 versus control group; and #p < 0.05 versus diabetic group. (C) The graph demonstrates the levels of SOCS3 (arbitrary units) per group. CB: 0.92 ± 0.05, Ex-4: 0.94 ± 0.08, D: 1.55 ± 0.07,
22
D+Ex-4: 0.63 ± 0.05, ***p < 0.001 versus control group; and ###p < 0.001 versus diabetic group. (D) The graph demonstrates the levels of p-AKT (Ser473) (arbitrary units) per group. CB: 0.65 ± 0.07, Ex-4: 0.51 ± 0.08, D: 0.84 ± 0.14, D+Ex-4: 0.62 ± 0.11, #p < 0.05 versus diabetic group. Values are mean ± S.E.M. n=5 animals for each group. CB: Vehicle-injected group, Ex-4: Exendin-4-injected group; D: Diabetic group and D+Ex-4: Exendin-4-injected diabetic
A
CC
EP
TE
D
M
A
N
U
SC RI PT
group.
23
Table 1. List of antibodies used for immunocytochemistry and Western blotting. Dilution
Application
Company
Catalog number
PCNA
1:50
Immunohistochemistry
Thermo Scientific
RB-9055-P
Cleaved Caspase-3
1:100
Westen blotting
Millipore
AB3623
1:100
Immunohistochemistry
CST
Pro-SPC
1:5000
Western blotting
Millipore
AB3786
RAGE
1:500
Western blotting
Novus Biologicals
NBP-1-42612
α-SMA
1:500
Western blotting
Santa Cruz
sc-32251
Collagen
1:1000
Western blotting
Novus Biologicals
NBP-1-30054
Fibronectin
1:10000
Western blotting
BD Biosciences
BD-610077
TGF-β
1:500
Western blotting
p-IRS1
1:1000
Western blotting
SOCS3
1:1000
p-AKT
A
N
U
SC RI PT
Antibody
9661S
sc-146
CST
2381S
Western blotting
CST
2932S
1:1000
Western blotting
CST
4060-S
β-actin
1:500
Western blotting
Santa Cruz
sc-47778
Goat antimouse IgG
1:5000
Western blotting
Abcam
ab98679
Western blotting
Santa Cruz
sc-2004
EP
TE
D
M
Santa Cruz
1:5000
A
CC
Goat anti-rabbit IgG
24