- Email: [email protected]
Diabetes-accelerated experimental osteoarthritis is prevented by autophagy activation
Accepted Manuscript Diabetes-accelerated Experimental Osteoarthritis is Prevented by Autophagy Activation Madalena Ribeiro, BS, Paloma López de Figueroa, BS, Uxía Nogueira-Recalde, BS, Alberto Centeno, MD, Alexandrina F. Mendes, PhD, Francisco J. Blanco, MD, Beatriz Caramés, PhD PII:
S1063-4584(16)30170-4
DOI:
10.1016/j.joca.2016.06.019
Reference:
YJOCA 3793
To appear in:
Osteoarthritis and Cartilage
Received Date: 7 March 2016 Revised Date:
17 June 2016
Accepted Date: 27 June 2016
Please cite this article as: Ribeiro M, López de Figueroa P, Nogueira-Recalde U, Centeno A, Mendes AF, Blanco FJ, Caramés B, Diabetes-accelerated Experimental Osteoarthritis is Prevented by Autophagy Activation, Osteoarthritis and Cartilage (2016), doi: 10.1016/j.joca.2016.06.019. 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.
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Diabetes-accelerated Experimental Osteoarthritis is Prevented by Autophagy
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Activation Madalena Ribeiro1,3, BS, Paloma López de Figueroa1, BS, Uxía Nogueira-Recalde1,
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BS, Alberto Centeno2, MD, Alexandrina F. Mendes3, PhD, Francisco J. Blanco1, MD,
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Beatriz Caramés1*, PhD
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This study was supported by Instituto de Salud Carlos III-Ministerio de
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Economía y Competitividad, Spain-CP11/00095 and Fondo Europeo de Desarrollo
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Regional (FEDER). Madalena Ribeiro was supported by Fundo Social Europeu
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and Fundação Portuguesa para a Ciência e a Tecnologia (FCT), PhD fellowship,
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SFRH/BD/78188/2011. Beatriz Caramés was supported by Miguel Servet Program,
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Instituto de Salud Carlos III-CP11/00095. 1
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Madalena Ribeiro, Paloma López de Figueroa, Uxía Nogueira-Recalde,
Francisco J. Blanco and Beatriz Caramés: Unidad de Biología del Cartílago, Grupo de
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Reumatología. Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo
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Hospitalario Universitario de A Coruña (CHUAC), España. 2Alberto Centeno: Unidad
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de Cirugía Experimental, INIBIC-CHUAC,
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Mendes: Centre for Neuroscience and Cell Biology and Faculty of Pharmacy,
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University of Coimbra, Portugal.
Madalena Ribeiro, Alexandrina F.
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Address correspondence and reprint request to Beatriz Caramés, PhD, Unidad de
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Biología del Cartílago, Grupo de Reumatología. Instituto de Investigación Biomédica
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de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC),
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As Xubias 84 15006-A Coruña, SPAIN. Phone: +34-981176399, Fax: +34-981176398,
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E-mail: [email protected]
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Running Title: Autophagy in Diabetes-accelerated Joint Damage
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Keywords: Osteoarthritis, Diabetes, Cartilage, Synovium, Autophagy, mTOR 1
ACCEPTED MANUSCRIPT ABSTRACT
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Objective: Type 2 Diabetes (T2D) is a risk factor for Osteoarthritis (OA). Autophagy,
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an essential homeostasis mechanism in articular cartilage, is defective in T2D and OA.
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However, how T2D may influence OA progression is still unknown. We aimed to
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determine how diabetes affects cartilage integrity and whether pharmacological
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activation of autophagy has efficacy in diabetic mice with OA.
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Design: Experimental OA was performed in the right knee of 9 weeks-old C57Bl/6J
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male mice (Lean group, N=8) and of 9 weeks-old B6.BKS (D)-Leprdb male mice
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(db/db group, N=16) by transection of medial meniscotibial and medial collateral
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ligaments. Left knee was employed as control knee. Rapamycin (2 mg/kg weight/day)
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or Vehicle (dimethyl sulphoxide) were administered intraperitoneally 3 times a week for
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10 weeks. Histopathology of articular cartilage and synovium was evaluated by using
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semiquantitative
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Immunohistochemistry was employed to evaluate the effect of diabetes and Rapamycin
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on cartilage integrity and OA biomarkers.
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Results: Cartilage damage was increased in db/db mice compared to lean mice after
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experimental OA, while no differences are observed in the control knee. Cartilage
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damage and synovium inflammation were reduced by Rapamycin treatment of OA-
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db/db mice. This protection was accompanied with a decrease in MMP-13 expression
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and decreased IL-12 levels. Furthermore, autophagy was increased and cartilage
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cellularity was maintained, suggesting that mTOR targeting prevents joint physical
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harm.
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Conclusion: Our findings indicate that diabetic mice exhibit increased joint damage
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after experimental OA, and that autophagy activation might be an effective therapy for
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diabetes-accelerated OA.
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INTRODUCTION Osteoarthritis (OA) is the most common joint disease and represents an enormous socioeconomic burden affecting millions of people worldwide (1). Type 2 Diabetes (T2D) is a multifactorial chronic metabolic disease that results
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from defects in insulin secretion and/or action, resulting in hyperglycemia,
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hyperinsulinemia, relative β-cell dysfunction and insulin resistance, which is
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characterized by the failure of tissues to respond appropriately to insulin (2).
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As age-related diseases, both OA and T2D are expected to rise due to increase of
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life expectancy (3).
In OA, cartilage and many of its surrounding tissues are disrupted. Patients often
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experience pain, stiffness and loss of mobility, which leads to a functional impairment,
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incapacity and disability in elderly people (4). With the increase of medical and
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disability costs and in the absence of an effective treatment for preventing OA, a better
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delineation of the risk factors becomes essential to investigate the cellular signaling
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pathways underlying the disease. This understanding will provide opportunities to
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identify new therapeutic targets that might predict OA severity and prevent progression.
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Epidemiological and experimental reports suggest a positive association between
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OA and T2D, however, there is no comprehensive explanation for this association and
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further studies are required to understand the mechanism by which T2D accelerates OA
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progression (5). T2D could be an important risk factor for OA (6,7) by affecting
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cartilage homeostasis, accelerating OA progression and altering the prognosis by
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increasing the risk of total joint replacement (8-10). Thus, T2D patients seem to have an
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increased susceptibility to develop OA, which is independent of age, sex and body
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weight (10).
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patients showed reduced autophagy, an essential cartilage homeostasis mechanism,
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which is associated to increased mTOR activity compared to chondrocytes from non-
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diabetic OA patients. These results suggest that defective autophagy might be one of the
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mechanisms responsible for the cartilage degradation observed in diabetic patients (11).
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Autophagy is an intracellular mechanism that contributes to maintain cellular
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integrity, function and survival by the clearance of unnecessary/damaged proteins and
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organelles (12). This mechanism is very important in poorly irrigated post-mitotic
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tissues, such as articular cartilage, with a very low rate of cell turnover and little
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regenerative capacity (13). Autophagy dysfunction contributes to the pathogenesis of
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many human diseases, including T2D and OA (14). Non-autonomous regulation of
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autophagy is observed in metabolic tissues relevant in T2D such as pancreas and
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adipose tissue (15-19). In chondrocytes from OA patients, we demonstrated that
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autophagy markers are decreased, suggesting that compromised autophagy capacity
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could contribute to the development of OA (20).
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Autophagy is regulated by multiple signaling pathways, most of them
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converging on mammalian target of rapamycin (mTOR). mTOR is a serine/threonine
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kinase that regulates cell growth, protein synthesis and metabolism, and it is considered
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a major negative regulator of autophagy (21). mTOR plays an important role in age-
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related diseases including T2D and OA. Chronic activation of mTOR by growth factors
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and amino acids cause insulin resistance (22,23), indicating that dysregulation of mTOR
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pathway is intimately associated with diabetes (24). mTOR plays an important role in
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cartilage homeostasis, contributing to the degenerative process involved in OA. In fact,
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mTOR is overexpressed in human OA cartilage (25), while mTOR inhibition prevents
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OA progression. In experimental OA performed in mice, both genetic deletion of
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mTOR and pharmacological inhibition of mTOR by Rapamycin lead to reduced OA
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severity (25-28). In the recent years the interaction between OA and T2D has been increasingly
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studied. However, lack of in vivo studies hampered the definition of mechanisms
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involved in the consequences of metabolic disease for OA. Thus, the objective of this
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study was to determine how diabetes affects cartilage integrity and to evaluate the
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efficacy of pharmacological activation of autophagy to reduce experimental OA in
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diabetic mice.
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METHOD
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Animal studies Eight-week-old male db/db mice (B6.BKS (D)-Leprdb/J; JAX stock 000697)
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and the respective background strain mice (C57BL/6J-non-diabetic lean mice; JAX
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stock 000664) were purchased from The Jackson Laboratory. Db/db mouse, originated
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from C57BL/6J is one of the best characterized Type 2 diabetes (T2D) animal model.
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The db/db mouse lacks the leptin receptor due to an spontaneous autosomal recessive
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mutation. This mouse becomes obese around 3 to 4 weeks of age, with elevations of
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plasma insulin beginning at 10 to 14 days and of blood sugar at 4 to 8 weeks. Mice were
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housed in a 12h light/dark cycle and temperature-controlled environment, with ad
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libtium access to food and water. Animal experiments were conducted in accordance
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with the guidelines of the Institutional Animal Care and Use Committee of Biomedical
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Research Institute (INIBIC) and in accordance with the ARRIVE (Animal Research:
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Reporting of In Vivo Experiments) guidelines.
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Experimental Groups and randomization
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Lean mice (C57BL/6J-Non-diabetic mice;N=8); db/db-Vehicle mice (N=8) and
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db/db-Rapamycin mice (N=8). Db/db mice were randomized according to body weight
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and blood glucose levels in two groups of N=8. At the beginning of the study the
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differences of body weight and blood glucose levels were not significantly different
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between both groups.
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Experimental Osteoarthritis
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Following acclimation for one week, all animals were anesthesized with
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sevofurane and experimental OA was induced in nine weeks-old male mice by
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(MMTL+MCL) in the right knee by a trained veterinary surgeon. The left knee was not
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subjected to surgery and was used as a control (26). All mice were allowed to move
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freely within their cages after surgery. Recovery was monitorized and a clinical follow-
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up was performed in the following days. 10 weeks after the knee surgery, the animals
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were fasted for 16 hours and then euthanized by CO2 inhalation.
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Rapamycin treatment
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Rapamycin purchased from LC Laboratories (Woburn, MA), was dissolved in
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Dimethyl Sufoxide (DMSO) at 25 mg/ml and stored at -20ºC. The stock solution was
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diluted in phosphate buffered saline (PBS) for injection. Treatment was started the day
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after the surgery and continued for 10 weeks. Rapamycin was injected intraperitoneally,
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three days per week at 2 mg/kg body weight/dose in a total injection volume of 0.2ml
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for 10 weeks. Importanly, the drug administration was well tolerated by all animals. The
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control group received the DMSO vehicle in PBS. The body weight was monitored
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weekly to adjust the dose of Rapamycin/Vehicle to inject. The dosage and frequency of
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Rapamycin administration were selected based on previous studies (26, 29).
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Glucose determination
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For glucose measurement, mice were fasted for 16 h, and blood was collected
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from the tail vein. Fasted glucose levels were monitored every two weeks. Blood
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glucose was measured using a FreeStyle Freedom Lite Glucometer (Abbott).
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Quantification of Insulin
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Blood samples were collected from sacrificed mice by cardiac puncture and
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plasma was separated using BD Microtrainer PST tubes with Lithium Heparin. Insulin
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levels were determined in plasma samples from all groups at the end of the study, using
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an Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc, USA).
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Liver Histology Liver tissues were fixed in formalin, dehydrated and embedded in paraffin, and
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4-µm-thick sections were stained with Hematoxylin-Eosin staining (H&E).
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Protein isolation and western blotting
Liver from db/db-Vehicle mice and db/db-Rapamycin mice were collected at the
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time of sacrifice and was immediately frozen in liquid nitrogen and stored at -80ºC until
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use. Liver was pulverized in a liquid nitrogen-cooled freezer (Microdismembrator 200,
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Retsch®) at the rate of maximum impact frequency (25Hz, 5 minutes). The proteins
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were resuspended in lysis buffer (6M urea/2%SDS buffer) and mixed for 1 hour at room
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temperature in an orbital shaker. The homogenate was centrifuge and the supernatant
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was recovered. The protein concentrations in the supernatant were determined using a
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bicinchoninic acid reagent assay (BCA). The concentrated samples were then adjusted
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to equal volumes before resolution on 4–20% gels (Invitrogen). Protein was transferred
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onto nitrocellulose membranes (Invitrogen) and then, blocked with 5% dry milk in Tris
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buffered saline–Tween (TBST). The membranes were incubated with antibodies for
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phospho-AMPK (Thr172) (1:1000) and phospho-rpS6 (Ser 235/236) (1:2000) (Cell
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Signaling Technology, Beverly, MA; 2535, 4858, respectively), GAPDH (1:10000,
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Invitrogen) and α-tubulin (1:2000, Sigma; T9026). Then, the membranes were
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incubated with horseradish peroxidase–conjugated anti-mouse IgG (Cell Signaling
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Technology) for 1 hour. Afterward, the membranes were washed 3 times with TBST
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and developed using an enhanced chemiluminescent substrate from Pierce. The
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intensity of the bands was analyzed by using TotalLab (TL120) software.
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Histological analysis of mouse knee joints Mice knee joints from the three groups were harvested. The joints were fixed in
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formalin for 24 hours, decalcified solution (Decalc™, HistoLab, 00601) for 6 hours, and
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then embedded in paraffin. Serial sagittal sections (4µm) were cut, stained with Safranin
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O-fast green, and examined for histopathological changes using the OARSI
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semiquantitative scoring system (30). In this system the scores are defined as follows:
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0=normal cartilage, 0.5=loss of proteoglycan with an intact surface, 1=superficial
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fibrillation without loss of cartilage, 2=vertical clefts and los surface lamina (any % or
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joint surface area), 3=vertical clefts/ erosion to the calcified layer lesion for 1–25% of
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the quadrant width, 4=lesion reaches the calcified cartilage for 25–50% of the quadrant
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width, 5=lesion reaches the calcified cartilage for 50–75% of the quadrant width,
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6=lesion reaches the calcified cartilage for >75% of the quadrant width.
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Histological analysis of inflammation
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Synovial inflammation was examined using a specific grading system (31).
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Three parameters, hyperplasia/enlargement of synovial lining cell layer, activation of
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resident cells/synovial stroma and inflammatory cell infiltration were graded as absent
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(grade 0), slight (grade 1), moderate (grade 2) or severe (grade 3).
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Immunohistochemistry
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Sections from paraffin-embedded joints were first deparaffinized in the xylene
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and rehydrated in graded ethanol and water. For antigen unmasking, sections in 10 mM
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sodium citrate buffer (pH 6.0) were heated in a microwave oven and kept at 80–85°C
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for 1.5 minutes. Slides were cooled for 20 minutes at room temperature after antigen
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unmasking. After washing with phosphate buffered saline (PBS), sections were blocked
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with 5% serum for 30 minutes at room temperature. Then, sections were incubated with 10
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(Cell Signaling Technology, Beverly, MA; 3868) overnight at 4°C. After washing with
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PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody for
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30 minutes at room temperature, and then incubated using Vectastain ABC-AP alkaline
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phosphatase (Vector Laboratories, Burlingame, CA) for 30 minutes. Slides were
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washed, and sections were incubated with 3,3´-diaminobenzidine (DAB) substrate for
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3-10 minutes.
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Quantification of MMP-13-positive cells.
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In synovium from Lean, db/db-Vehicle and db/db-Rapamycin, the total number
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number of MMP-13-positive cells was counted in two independent areas of each
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section.
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Mouse Cytokine Magnetic 10-Plex Panel
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was separated using BD Microtrainer PST tubes with Lithium Heparin. Cytokines levels
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were determined in serum samples using a Mouse Cytokine Magnetic 10-Plex Panel for
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simultaneous quantitative determination of GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-
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6, IL-10, IL-12p70 and TNF-α (Invitrogen; LMC0001M).
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Cartilage cellularity
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Knee joint sections were stained with Hematoxylin and Eosin (H&E). In
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cartilage from knees with experimental OA, three pictures were taken under 10x
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magnification, representing the centre of the femoral condyle that is not covered by the
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menisci as well as the medial and lateral femoral condyles. The total number of cells in
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each section was counted (32).
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Statistical analysis To test for normal distribution of the data, we used the Kolmogorov-Smirnov
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test. In general, the data sets follow a normal distribution. In the cases where the
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number of samples was too small to be tested accurately, we assumed the data follow a
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normal distribution. Statistically significant differences between 2 groups were
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determined by Student's t-test. Statistically significant differences between multiple
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groups were determined analyzing variance (ANOVA) in conjunction with multiple
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comparison test. Data are presented as the mean (lower level, upper level) (ll, ul) 95%
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confidence interval (CI). The data analysis and statistical inference was performed by
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using Prism 5.0 software (GraphPad Software, La Jolla, CA, USA).
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RESULTS
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Autophagy activation by Rapamycin improves metabolic parameters in db/db
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mice Diabetic (db/db) mice are obese, hyperglycemic and hyperinsulinemic with
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markedly insulin resistant. At the end of our study, body weight of db/db mice was 32%
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higher than Lean mice (47,5±1,254 g vs 32,25±0,70 g, p<0.0001). Importantly,
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Rapamycin treatment significantly reduced body weight in db/db mice (Figure 1A).
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Furthermore, a marked increase in fasted blood glucose levels and plasma insulin levels
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were observed in db/db mice compared to Lean group. However, db/db mice subjected
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to experimental OA and treated with Rapamycin exhibited a significant reduction in
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fasted blood glucose levels and plasma insulin levels, indicating that Rapamycin
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treatment improves metabolic parameters in db/db mice (Figure 1B). In addition, a
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dramatic reduction in lipid accumulation is observed in liver tissue of Rapamycin
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treated animals (Figure 1C). To demonstrate target engagement of Rapamycin
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treatment, phosphorylation of AMP activated protein Kinase (p-AMPK) and
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phosphorylation of ribosomal protein S6 (p-prS6), a direct target of mTOR, were
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determined. Indeed, Rapamycin significantly increased p-AMPK expression and
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significantly reduced p-prS6 expression in liver tissue (Figure 1D).
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Diabetes accelerates experimental OA in db/db mice
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To investigate the influence of T2D in OA progression, Lean mice and db/db
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mice were subjected to experimental OA by transection of the medial meniscotibial
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ligament and the medial collateral ligament in the right knee. Left knee joints were used
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as an internal control. 10 weeks after surgery, right and left knee joints were evaluated
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Lean and db/db mice (Figure 2 A,B). A significant increase in cartilage damage was
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found in OA-db/db mice compared to OA-Lean mice (Figure 2C), while control knee
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joints did not show histopathological cartilage changes. These results suggest first, that
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diabetes accelerates cartilage damage after experimental OA and second, that this effect
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is associated to metabolic disturbances of diabetic mice.
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mTOR inhibition prevents diabetes-accelerated experimental OA
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To evaluate if pharmacological mTOR inhibition has beneficial effects on
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diabetes-accelerated OA, db/db mice were subjected to experimental OA (N=16).
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Db/db-Vehicle mice exhibited OA-like changes with loss of proteoglycans and
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structural changes in the surface lamina. On the other hand, db/db-Rapamycin mice only
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showed a focal loss of proteoglycans and small structural changes in the articular
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cartilage (Figure 2A,B), associated to a reduced histological OA score (Figure 2C).
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These
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accelerated experimental OA in mice.
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Rapamycin reduces synovium inflammation in diabetes-accelerated experimental
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OA
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Inflammation is important in OA and T2D pathophysiology (33), although how
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bioenergy sensors link metabolism with inflammatory processes to regulate joint
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integrity is unknown. We investigated how diabetes affects the synovium of db/db mice.
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In this sense, a significant increase of synovial inflammation is observed after surgery in
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both Lean and db/db mice, although a greater inflammation score is found in db/db
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mice. Interestingly, Rapamycin treatment reduced synovial inflammation in db/db while
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no signatures of histopathological changes were observed in the Control knee joints
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(Figure 3A,B). These results suggest that the beneficial effect of mTOR inhibition in
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joints might be mediated by a reduction in synovium inflammation.
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Rapamycin reduces the expression of catabolic factors involved in cartilage loss
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and inflammation in db/db mice
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To evaluate the mechanism of action of diabetes-accelerated cartilage
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degradation in db/db mice, immunohistochemistry analysis was performed for MMP-
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13, an important OA biomarker involved in cartilage degradation (34). The results
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showed a significant increase in the expression of MMP-13 in OA db/db-Vehicle mice,
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predominantly in the synovium area. Importantly, Rapamycin treated OA db/db mice
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showed a significant reduction in the expression of MMP-13, indicating a potent anti-
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catabolic effect of mTOR inhibition in db/db mice (Figure 4A,B).
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To go further in the explanation of the protective effects of Rapamycin,
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proinflammatory cytokine levels were evaluated in plasma from db/db-Vehicle mice
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and db/db-Rapamycin mice. The results showed a significant reduction in serum levels
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of active interleukin 12 (IL-12p70), a proinflammatory cytokine (35), in db/db-
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Rapamycin mice compared to db/db-Vehicle mice (p<0.01) (Figure 4C), suggesting a
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potential common link between T2D and OA inflammatory mechanisms.
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Rapamycin maintains chondrocyte homeostasis in db/db mice
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Autophagy, a key homeostatic mechanism in articular cartilage, is reduced in
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chondrocytes from OA diabetic patients (11). To confirm this evidence, LC3, a main
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marker for autophagosome formation (36), was evaluated. The results showed that LC3
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staining was reduced after experimental OA in db/db-Vehicle mice. Remarkably,
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mTOR inhibition by Rapamycin maintains LC3 expression in db/db mice knee joints
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subjected to experimental OA (Figure 5A). Chondrocyte cellularity in mouse knee joints was significantly reduced in
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chondrocytes in OA-db/db-Vehicle mice compared to OA-Lean mice. Preservation of
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cellularity is observed in Rapamycin treated mice compared to OA db/db-Vehicle mice
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(Figure 5B).
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Together, these data suggest that cartilage homeostasis is compromised in db/db
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chondrocyte loss.
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DISCUSSION Metabolism influences dramatically health outcomes, including articular
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cartilage and synovium function. However, the consequences of metabolic
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complications of diabetes in OA are not well understood. There are evidences indicating
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that failure of homeostasis mechanisms such as autophagy, leads to alterations in
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articular cartilage that ultimately cause joint damage and OA (20). Based on these
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concepts, we previously demonstrated that high glucose and high insulin reduced
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autophagy in human chondrocytes and accelerated cartilage degradation through
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regulation of Akt/mTOR signaling pathway. We found that pharmacological activation
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of autophagy attenuates the catabolic effects of high glucose and high insulin in human
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chondrocytes. Our findings suggest that reduced autophagy might be one of the
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mechanisms responsible for the cartilage degradation observed in T2D patients (11).
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In the present study, we investigate the implication of T2D in OA development
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by using a genetic diabetes mice model. These mice have a mutation on the leptin
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receptor and lack leptin signaling, which leads to hyperphagia, persistent
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hyperinsulinemia, hyperglycemia and obesity (37). Db/db mice exhibit metabolic
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disturbances similar to those observed in humans with T2D, being a representive model
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to study diabetes and the associated metabolic complications (38). Human metabolic
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diseases have been linked to mTOR dysregulation (39), We evaluated how systemic
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Rapamycin treatment, an autophagy inducer by mTORC1 inhibition, influences
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metabolic phenotype of db/db mice. Rapamycin significantly improved body weight
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gain, fasted blood glucose levels and serum insulin levels in db/db mice. To investigate
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the link with the targets potentially responsible for these beneficial effects, we evaluated
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the activity of AMPK, a key energy sensor that regulates cellular metabolism to
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ACCEPTED MANUSCRIPT maintain energy homeostasis and p-rpS6, a direct target of mTOR (40). Consistent with
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established downstream activity, Rapamycin reduced p-rpS6 while increased the
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phosphorylation of AMPK. The data is a confirmation of the pharmacological activity
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of Rapamycin in our model, related to AMPK response to energy stress probably
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through inhibition of the mTORC1 pathway and autophagy activation (41).
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To study the connection between T2D and OA progression, we performed
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experimental OA in Lean and db/db mice. Our results showed that articular cartilage of
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OA-db/db mice developed more severe OA-like changes in articular cartilage compared
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to OA-Lean mice. Notably, synovium inflammation and thickening was found in the
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joints of OA-db/db mice. Joint inflammation was accompanied with an increase in
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MMP-13, predominantly in the synovial membrane. Together, these results support the
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concept that abnormal glucose metabolism, primarily associated with high levels of
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glucose and insulin accelerates experimental OA in db/db mice.
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The maintenance of autophagy is essential to preserve cartilage integrity.
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Decreased autophagy and increased chondrocyte death now established features of OA
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(42,43). In this sense, articular cartilage from diabetic mice displayed a reduction of
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LC3, and a decrease in cartilage cellularity, which suggest that joint damage observed in
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db/db mice might be due to defective autophagy which leads to an imbalance between
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the catabolic and anabolic pathways.
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Since decreased in autophagy could be one of the mechanisms responsible for
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the faster cartilage damage observed in diabetes, we evaluated whether pharmacological
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autophagy activation by Rapamycin could confer protection to joints. Indeed,
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Rapamycin treatment reduces the severity of experimental OA in C57BL/6J mice and
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prevents OA progression, consistent with previous observations (25-28). Our results
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show that Rapamycin attenuates the severity of experimental OA in db/db mice,
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Rapamycin treatment dramatically reduces synovial inflammation, the expression of
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MMP-13 and circulating IL-12 levels were significantly reduced in response to
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Rapamycin. Previous studies demonstrated an increase in IL-12 levels in serum from
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patients with T2D (44) and in mice subjected to high fat diet and posttraumatic arthritis
434
(45), suggesting a potential common inflammatory pathway for diabetes and OA (46).
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In fact, OA cartilage from diabetic patients is more reactive to pro-inflammatory stress
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than OA cartilage from non-diabetic patients, suggesting that diabetes increases
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inflammation in OA (47). Our results are consistent with previous findings where
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autophagy activation can prevent inflammatory responses (48,49).
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Furthermore, Rapamycin increases LC3 expression and maintains the
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chondrocyte number in db/db mice. These findings together with our previous
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observations suggest that Rapamycin treatment reduces joint damage in diabetic mice
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with experimental OA by both normalizing altered metabolic parameters of T2D and by
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inducing autophagy.
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It is important to note that db/db mice are not only diabetic, but they have
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increased body weight and obesity, which is also an important risk factor for OA (37,
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45). Since we performed experimental OA only in right knee, left knee was employed as
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a control to evaluate the direct effect of obesity in the joint in this time frame. We
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observed increased cartilage damage and synovial inflammation in the OA knee, while
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control knee did not show any pathological changes. These results suggest that body
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weight is not sufficient to accelerate experimental OA in db/db mice. One of the
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limitations of this study is that contralateral limbs of DMM were used as control joints
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as opposed to sham surgery joints. It is possible that an alteration of joint load can be
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developed as a result of experimental OA in the joint.
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To conclude, our study identifies a promising therapeutic approach to prevent
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cartilage degradation observed in T2D patients. However, Rapamycin, has some effects,
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which limits its use for preventive strategies in humans (50). Further studies are needed
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to identify new drugs that can activates autophagy and modify the OA progression in
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diabetic patients. This knowledge could leads to an improvement of clinical care in OA
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patients with metabolic diseases.
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ACCEPTED MANUSCRIPT Acknowledgements
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We thank the personal of the Experimental Unit for the animal maintenance.
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Purificación Filgueira and Noa Goyanes from the Histology Core and Lucia Lourido for
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technical assistance.
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Author contributions
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All authors approved the final version to be published.
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Dra. Caramés had full access to all of the data in the study and takes responsibility for
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the integrity of the data and the accuracy of the data analysis.
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Study conception and design. Mendes, Blanco, Caramés.
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Drafting and revising the manuscript. Ribeiro, López de Figueroa, Nogueira-Recalde,
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Centeno, Mendes, Blanco, Caramés.
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Acquisition of data. Ribeiro, López de Figueroa, Nogueira-Recalde, Caramés.
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Analysis and interpretation of data. Ribeiro, Caramés.
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Role of the Funding Source
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This study was supported by Instituto de Salud Carlos III-Ministerio de Economía y
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Competitividad, Spain-CP11/00095. Madalena Ribeiro was supported by Fundo Social
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Europeu (FSE), through "Programa Operacional Potencial Humano" (POPH),
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and national funds via FCT – Fundação Portuguesa para a Ciência e a Tecnologia under
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the PhD fellowship, SFRH/BD/78188/2011 and Beatriz Caramés was supported by
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Miguel Servet Program, Instituto de Salud Carlos III-CP11/00095.
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Competing interest
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The authors declare that there is no conflict of interest regarding the publication of this
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paper.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
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Figure 1. Autophagy activation by Rapamycin improves metabolic parameters in
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db/db mice. A, Body weight of Lean, db/db-Vehicle and db/db-Rapamycin mice was
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evaluated every week during 10 weeks. Values are the means (ll, ul) 95% CI of eight
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mice in each experimental group. *p<0.0001 vs. Lean mice and **p (t0:p=0.4482,
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t1:p=0.2376, t2=0.0673, t3:p=0.0382, t4:p=0.0117, t5:p=0.0004, t6:p<0.0001, t7:p=0.0030,
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t8:p=0.0009, t9:p=0.0003, t10:p=0.0002) vs. db/db-Vehicle mice. B, Fasted blood
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glucose levels were determined after three measurements during the study. Values are
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the means (ll, ul) 95% CI of eight mice in each experimental group. Fasted plasma
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insulin levels were determined at the end of the study. Values are the means (ll, ul) 95%
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CI of eight mice in each experimental group. C, Liver sections from db/db-Vehicle and
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db/db-Rapamycin mice (N=8 each/group) were analyzed by staining with H&E.
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Representative images of liver sections from two mice per group. D, Liver protein
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extracts from db/db-Vehicle mice and db/db-Rapamycin mice. Phosphorylation of AMP
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activated protein kinase (AMPK) and ribosomal protein S6 (rpS6) was determined by
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Western blotting. Densitometric analysis of p-AMPK and p-rpS6. GAPDH and α-
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Tubulin were employed as a loading control. Values are the means (ll, ul) 95% CI of 5
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mice in each experimental group.
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Figure 2. Diabetes-accelerated experimental osteoarthritis is prevented by mTOR
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inhibition. Nine-week-old male db/db mice (B6.BKS (D)-Leprdb/J, N=16) and the
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respective background strain mice (C57BL/6J-non-diabetic lean mice, N=8) were
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subjected to experimental osteoarthritis (OA) in the right knee (OA). The left knee was
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not subjected to surgery and was used as a control (Ctrl). Three experimental mice
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groups were performed; Lean mice, db/db-Vehicle mice and db/db-Rapamycin mice.
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Each experimental group included eight mice. A-B, Knee joints were analyzed by
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histological scores of Lean, db/db-Vehicle and db/db-Rapamycin mice. Values are the
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means (ll, ul) 95% CI of eight mice in each experimental group (each point represents
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an individual mice).
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Figure 3. Effect of Rapamycin on synovial inflammation in experimental OA. A,
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Synovium from Lean, db/db-Vehicle and db/db-Rapamycin mice (Ctrl and OA, N=8
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each group) was analysed by staining with Hematoxylin Eosin (H&E). Scale bar
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100µm. Magnification: 10x. B, The histological score for inflammation was
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significantly increased in OA-db/db-Vehicle mice compared to OA-Lean mice and
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significantly decreased after Rapamycin treatment. Values are the means (ll, ul) 95% CI
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of eight mice in each experimental group (each point represents an individual mice).
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Figure 4. Rapamycin reduces the expression of catabolic factors implicated in the
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joint damage in db/db mice. A, Knee joints from Lean, db/db-Vehicle and db/db-
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Rapamycin mice were collected 10 weeks after surgery (N=5 each/group). Sections
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were analysed by immunohistochemistry for MMP-13. Two sections from one animal
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are presented. Scale bar 100µm. Magnification: 10x. B, Quantitative analysis of MMP-
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13 positive cells. Values are the means (ll, ul) 95% CI of five mice in each experimental
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group (each point represents the mean of two independent areas from each mice). C,
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Quantitative analysis of Interleukin-12 (IL12p70) in serum from db/db-Vehicle and
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db/db-Rapamycin mice. Values are the means (ll, ul) 95% CI of six mice in db/db-
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Vehicle group and eight mice in db/db-Rapamycin group (each point represents
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individual mice).
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Figure 5. Rapamycin maintains chondrocyte homeostasis in db/db mice. A, Knee
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joint from Lean, db/db-Vehicle and db/db-Rapamycin mice with experimental OA (N=3
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autophagosome formation and autophagy activation. Scale bar 100µm. Magnification:
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10x. B, Quantitative analysis of cell number in OA-Lean, OA-db/db-Vehicle and OA-
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db/db-Rapamycin mice (N=5 each/group). Values are the means (ll, ul) 95% CI of five
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mice in each experimental group (each point represents the mean of three independent
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areas for each mice).
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S1063-4584(16)30170-4
DOI:
10.1016/j.joca.2016.06.019
Reference:
YJOCA 3793
To appear in:
Osteoarthritis and Cartilage
Received Date: 7 March 2016 Revised Date:
17 June 2016
Accepted Date: 27 June 2016
Please cite this article as: Ribeiro M, López de Figueroa P, Nogueira-Recalde U, Centeno A, Mendes AF, Blanco FJ, Caramés B, Diabetes-accelerated Experimental Osteoarthritis is Prevented by Autophagy Activation, Osteoarthritis and Cartilage (2016), doi: 10.1016/j.joca.2016.06.019. 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.
ACCEPTED MANUSCRIPT 1
Diabetes-accelerated Experimental Osteoarthritis is Prevented by Autophagy
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Activation Madalena Ribeiro1,3, BS, Paloma López de Figueroa1, BS, Uxía Nogueira-Recalde1,
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BS, Alberto Centeno2, MD, Alexandrina F. Mendes3, PhD, Francisco J. Blanco1, MD,
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Beatriz Caramés1*, PhD
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This study was supported by Instituto de Salud Carlos III-Ministerio de
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Economía y Competitividad, Spain-CP11/00095 and Fondo Europeo de Desarrollo
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Regional (FEDER). Madalena Ribeiro was supported by Fundo Social Europeu
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and Fundação Portuguesa para a Ciência e a Tecnologia (FCT), PhD fellowship,
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SFRH/BD/78188/2011. Beatriz Caramés was supported by Miguel Servet Program,
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Instituto de Salud Carlos III-CP11/00095. 1
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Madalena Ribeiro, Paloma López de Figueroa, Uxía Nogueira-Recalde,
Francisco J. Blanco and Beatriz Caramés: Unidad de Biología del Cartílago, Grupo de
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Reumatología. Instituto de Investigación Biomédica de A Coruña (INIBIC), Complexo
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Hospitalario Universitario de A Coruña (CHUAC), España. 2Alberto Centeno: Unidad
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de Cirugía Experimental, INIBIC-CHUAC,
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Mendes: Centre for Neuroscience and Cell Biology and Faculty of Pharmacy,
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University of Coimbra, Portugal.
Madalena Ribeiro, Alexandrina F.
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Address correspondence and reprint request to Beatriz Caramés, PhD, Unidad de
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Biología del Cartílago, Grupo de Reumatología. Instituto de Investigación Biomédica
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de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC),
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As Xubias 84 15006-A Coruña, SPAIN. Phone: +34-981176399, Fax: +34-981176398,
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E-mail: [email protected]
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Running Title: Autophagy in Diabetes-accelerated Joint Damage
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Keywords: Osteoarthritis, Diabetes, Cartilage, Synovium, Autophagy, mTOR 1
ACCEPTED MANUSCRIPT ABSTRACT
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Objective: Type 2 Diabetes (T2D) is a risk factor for Osteoarthritis (OA). Autophagy,
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an essential homeostasis mechanism in articular cartilage, is defective in T2D and OA.
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However, how T2D may influence OA progression is still unknown. We aimed to
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determine how diabetes affects cartilage integrity and whether pharmacological
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activation of autophagy has efficacy in diabetic mice with OA.
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Design: Experimental OA was performed in the right knee of 9 weeks-old C57Bl/6J
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male mice (Lean group, N=8) and of 9 weeks-old B6.BKS (D)-Leprdb male mice
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(db/db group, N=16) by transection of medial meniscotibial and medial collateral
35
ligaments. Left knee was employed as control knee. Rapamycin (2 mg/kg weight/day)
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or Vehicle (dimethyl sulphoxide) were administered intraperitoneally 3 times a week for
37
10 weeks. Histopathology of articular cartilage and synovium was evaluated by using
38
semiquantitative
39
Immunohistochemistry was employed to evaluate the effect of diabetes and Rapamycin
40
on cartilage integrity and OA biomarkers.
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Results: Cartilage damage was increased in db/db mice compared to lean mice after
42
experimental OA, while no differences are observed in the control knee. Cartilage
43
damage and synovium inflammation were reduced by Rapamycin treatment of OA-
44
db/db mice. This protection was accompanied with a decrease in MMP-13 expression
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and decreased IL-12 levels. Furthermore, autophagy was increased and cartilage
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cellularity was maintained, suggesting that mTOR targeting prevents joint physical
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harm.
and
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Conclusion: Our findings indicate that diabetic mice exhibit increased joint damage
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after experimental OA, and that autophagy activation might be an effective therapy for
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diabetes-accelerated OA.
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INTRODUCTION Osteoarthritis (OA) is the most common joint disease and represents an enormous socioeconomic burden affecting millions of people worldwide (1). Type 2 Diabetes (T2D) is a multifactorial chronic metabolic disease that results
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from defects in insulin secretion and/or action, resulting in hyperglycemia,
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hyperinsulinemia, relative β-cell dysfunction and insulin resistance, which is
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characterized by the failure of tissues to respond appropriately to insulin (2).
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As age-related diseases, both OA and T2D are expected to rise due to increase of
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life expectancy (3).
In OA, cartilage and many of its surrounding tissues are disrupted. Patients often
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experience pain, stiffness and loss of mobility, which leads to a functional impairment,
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incapacity and disability in elderly people (4). With the increase of medical and
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disability costs and in the absence of an effective treatment for preventing OA, a better
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delineation of the risk factors becomes essential to investigate the cellular signaling
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pathways underlying the disease. This understanding will provide opportunities to
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identify new therapeutic targets that might predict OA severity and prevent progression.
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Epidemiological and experimental reports suggest a positive association between
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OA and T2D, however, there is no comprehensive explanation for this association and
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further studies are required to understand the mechanism by which T2D accelerates OA
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progression (5). T2D could be an important risk factor for OA (6,7) by affecting
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cartilage homeostasis, accelerating OA progression and altering the prognosis by
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increasing the risk of total joint replacement (8-10). Thus, T2D patients seem to have an
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increased susceptibility to develop OA, which is independent of age, sex and body
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weight (10).
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patients showed reduced autophagy, an essential cartilage homeostasis mechanism,
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which is associated to increased mTOR activity compared to chondrocytes from non-
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diabetic OA patients. These results suggest that defective autophagy might be one of the
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mechanisms responsible for the cartilage degradation observed in diabetic patients (11).
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Autophagy is an intracellular mechanism that contributes to maintain cellular
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integrity, function and survival by the clearance of unnecessary/damaged proteins and
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organelles (12). This mechanism is very important in poorly irrigated post-mitotic
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tissues, such as articular cartilage, with a very low rate of cell turnover and little
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regenerative capacity (13). Autophagy dysfunction contributes to the pathogenesis of
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many human diseases, including T2D and OA (14). Non-autonomous regulation of
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autophagy is observed in metabolic tissues relevant in T2D such as pancreas and
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adipose tissue (15-19). In chondrocytes from OA patients, we demonstrated that
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autophagy markers are decreased, suggesting that compromised autophagy capacity
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could contribute to the development of OA (20).
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Autophagy is regulated by multiple signaling pathways, most of them
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converging on mammalian target of rapamycin (mTOR). mTOR is a serine/threonine
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kinase that regulates cell growth, protein synthesis and metabolism, and it is considered
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a major negative regulator of autophagy (21). mTOR plays an important role in age-
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related diseases including T2D and OA. Chronic activation of mTOR by growth factors
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and amino acids cause insulin resistance (22,23), indicating that dysregulation of mTOR
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pathway is intimately associated with diabetes (24). mTOR plays an important role in
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cartilage homeostasis, contributing to the degenerative process involved in OA. In fact,
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mTOR is overexpressed in human OA cartilage (25), while mTOR inhibition prevents
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OA progression. In experimental OA performed in mice, both genetic deletion of
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mTOR and pharmacological inhibition of mTOR by Rapamycin lead to reduced OA
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severity (25-28). In the recent years the interaction between OA and T2D has been increasingly
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studied. However, lack of in vivo studies hampered the definition of mechanisms
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involved in the consequences of metabolic disease for OA. Thus, the objective of this
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study was to determine how diabetes affects cartilage integrity and to evaluate the
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efficacy of pharmacological activation of autophagy to reduce experimental OA in
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diabetic mice.
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METHOD
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Animal studies Eight-week-old male db/db mice (B6.BKS (D)-Leprdb/J; JAX stock 000697)
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and the respective background strain mice (C57BL/6J-non-diabetic lean mice; JAX
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stock 000664) were purchased from The Jackson Laboratory. Db/db mouse, originated
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from C57BL/6J is one of the best characterized Type 2 diabetes (T2D) animal model.
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The db/db mouse lacks the leptin receptor due to an spontaneous autosomal recessive
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mutation. This mouse becomes obese around 3 to 4 weeks of age, with elevations of
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plasma insulin beginning at 10 to 14 days and of blood sugar at 4 to 8 weeks. Mice were
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housed in a 12h light/dark cycle and temperature-controlled environment, with ad
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libtium access to food and water. Animal experiments were conducted in accordance
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with the guidelines of the Institutional Animal Care and Use Committee of Biomedical
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Research Institute (INIBIC) and in accordance with the ARRIVE (Animal Research:
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Reporting of In Vivo Experiments) guidelines.
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Experimental Groups and randomization
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Lean mice (C57BL/6J-Non-diabetic mice;N=8); db/db-Vehicle mice (N=8) and
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db/db-Rapamycin mice (N=8). Db/db mice were randomized according to body weight
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and blood glucose levels in two groups of N=8. At the beginning of the study the
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differences of body weight and blood glucose levels were not significantly different
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between both groups.
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Experimental Osteoarthritis
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Following acclimation for one week, all animals were anesthesized with
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sevofurane and experimental OA was induced in nine weeks-old male mice by
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(MMTL+MCL) in the right knee by a trained veterinary surgeon. The left knee was not
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subjected to surgery and was used as a control (26). All mice were allowed to move
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freely within their cages after surgery. Recovery was monitorized and a clinical follow-
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up was performed in the following days. 10 weeks after the knee surgery, the animals
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were fasted for 16 hours and then euthanized by CO2 inhalation.
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Rapamycin treatment
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Rapamycin purchased from LC Laboratories (Woburn, MA), was dissolved in
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Dimethyl Sufoxide (DMSO) at 25 mg/ml and stored at -20ºC. The stock solution was
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diluted in phosphate buffered saline (PBS) for injection. Treatment was started the day
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after the surgery and continued for 10 weeks. Rapamycin was injected intraperitoneally,
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three days per week at 2 mg/kg body weight/dose in a total injection volume of 0.2ml
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for 10 weeks. Importanly, the drug administration was well tolerated by all animals. The
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control group received the DMSO vehicle in PBS. The body weight was monitored
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weekly to adjust the dose of Rapamycin/Vehicle to inject. The dosage and frequency of
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Rapamycin administration were selected based on previous studies (26, 29).
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Glucose determination
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For glucose measurement, mice were fasted for 16 h, and blood was collected
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from the tail vein. Fasted glucose levels were monitored every two weeks. Blood
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glucose was measured using a FreeStyle Freedom Lite Glucometer (Abbott).
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Quantification of Insulin
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Blood samples were collected from sacrificed mice by cardiac puncture and
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plasma was separated using BD Microtrainer PST tubes with Lithium Heparin. Insulin
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levels were determined in plasma samples from all groups at the end of the study, using
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an Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc, USA).
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Liver Histology Liver tissues were fixed in formalin, dehydrated and embedded in paraffin, and
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4-µm-thick sections were stained with Hematoxylin-Eosin staining (H&E).
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Protein isolation and western blotting
Liver from db/db-Vehicle mice and db/db-Rapamycin mice were collected at the
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time of sacrifice and was immediately frozen in liquid nitrogen and stored at -80ºC until
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use. Liver was pulverized in a liquid nitrogen-cooled freezer (Microdismembrator 200,
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Retsch®) at the rate of maximum impact frequency (25Hz, 5 minutes). The proteins
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were resuspended in lysis buffer (6M urea/2%SDS buffer) and mixed for 1 hour at room
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temperature in an orbital shaker. The homogenate was centrifuge and the supernatant
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was recovered. The protein concentrations in the supernatant were determined using a
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bicinchoninic acid reagent assay (BCA). The concentrated samples were then adjusted
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to equal volumes before resolution on 4–20% gels (Invitrogen). Protein was transferred
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onto nitrocellulose membranes (Invitrogen) and then, blocked with 5% dry milk in Tris
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buffered saline–Tween (TBST). The membranes were incubated with antibodies for
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phospho-AMPK (Thr172) (1:1000) and phospho-rpS6 (Ser 235/236) (1:2000) (Cell
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Signaling Technology, Beverly, MA; 2535, 4858, respectively), GAPDH (1:10000,
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Invitrogen) and α-tubulin (1:2000, Sigma; T9026). Then, the membranes were
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incubated with horseradish peroxidase–conjugated anti-mouse IgG (Cell Signaling
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Technology) for 1 hour. Afterward, the membranes were washed 3 times with TBST
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and developed using an enhanced chemiluminescent substrate from Pierce. The
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intensity of the bands was analyzed by using TotalLab (TL120) software.
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Histological analysis of mouse knee joints Mice knee joints from the three groups were harvested. The joints were fixed in
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formalin for 24 hours, decalcified solution (Decalc™, HistoLab, 00601) for 6 hours, and
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then embedded in paraffin. Serial sagittal sections (4µm) were cut, stained with Safranin
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O-fast green, and examined for histopathological changes using the OARSI
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semiquantitative scoring system (30). In this system the scores are defined as follows:
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0=normal cartilage, 0.5=loss of proteoglycan with an intact surface, 1=superficial
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fibrillation without loss of cartilage, 2=vertical clefts and los surface lamina (any % or
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joint surface area), 3=vertical clefts/ erosion to the calcified layer lesion for 1–25% of
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the quadrant width, 4=lesion reaches the calcified cartilage for 25–50% of the quadrant
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width, 5=lesion reaches the calcified cartilage for 50–75% of the quadrant width,
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6=lesion reaches the calcified cartilage for >75% of the quadrant width.
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Histological analysis of inflammation
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Synovial inflammation was examined using a specific grading system (31).
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Three parameters, hyperplasia/enlargement of synovial lining cell layer, activation of
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resident cells/synovial stroma and inflammatory cell infiltration were graded as absent
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(grade 0), slight (grade 1), moderate (grade 2) or severe (grade 3).
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Immunohistochemistry
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Sections from paraffin-embedded joints were first deparaffinized in the xylene
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and rehydrated in graded ethanol and water. For antigen unmasking, sections in 10 mM
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sodium citrate buffer (pH 6.0) were heated in a microwave oven and kept at 80–85°C
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for 1.5 minutes. Slides were cooled for 20 minutes at room temperature after antigen
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unmasking. After washing with phosphate buffered saline (PBS), sections were blocked
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with 5% serum for 30 minutes at room temperature. Then, sections were incubated with 10
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(Cell Signaling Technology, Beverly, MA; 3868) overnight at 4°C. After washing with
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PBS, sections were incubated with biotinylated goat anti-rabbit secondary antibody for
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30 minutes at room temperature, and then incubated using Vectastain ABC-AP alkaline
241
phosphatase (Vector Laboratories, Burlingame, CA) for 30 minutes. Slides were
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washed, and sections were incubated with 3,3´-diaminobenzidine (DAB) substrate for
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3-10 minutes.
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Quantification of MMP-13-positive cells.
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In synovium from Lean, db/db-Vehicle and db/db-Rapamycin, the total number
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number of MMP-13-positive cells was counted in two independent areas of each
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section.
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Mouse Cytokine Magnetic 10-Plex Panel
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was separated using BD Microtrainer PST tubes with Lithium Heparin. Cytokines levels
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were determined in serum samples using a Mouse Cytokine Magnetic 10-Plex Panel for
252
simultaneous quantitative determination of GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-
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6, IL-10, IL-12p70 and TNF-α (Invitrogen; LMC0001M).
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Cartilage cellularity
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Knee joint sections were stained with Hematoxylin and Eosin (H&E). In
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cartilage from knees with experimental OA, three pictures were taken under 10x
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magnification, representing the centre of the femoral condyle that is not covered by the
258
menisci as well as the medial and lateral femoral condyles. The total number of cells in
259
each section was counted (32).
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Statistical analysis To test for normal distribution of the data, we used the Kolmogorov-Smirnov
262
test. In general, the data sets follow a normal distribution. In the cases where the
263
number of samples was too small to be tested accurately, we assumed the data follow a
264
normal distribution. Statistically significant differences between 2 groups were
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determined by Student's t-test. Statistically significant differences between multiple
266
groups were determined analyzing variance (ANOVA) in conjunction with multiple
267
comparison test. Data are presented as the mean (lower level, upper level) (ll, ul) 95%
268
confidence interval (CI). The data analysis and statistical inference was performed by
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using Prism 5.0 software (GraphPad Software, La Jolla, CA, USA).
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RESULTS
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Autophagy activation by Rapamycin improves metabolic parameters in db/db
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mice Diabetic (db/db) mice are obese, hyperglycemic and hyperinsulinemic with
285
markedly insulin resistant. At the end of our study, body weight of db/db mice was 32%
286
higher than Lean mice (47,5±1,254 g vs 32,25±0,70 g, p<0.0001). Importantly,
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Rapamycin treatment significantly reduced body weight in db/db mice (Figure 1A).
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Furthermore, a marked increase in fasted blood glucose levels and plasma insulin levels
289
were observed in db/db mice compared to Lean group. However, db/db mice subjected
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to experimental OA and treated with Rapamycin exhibited a significant reduction in
291
fasted blood glucose levels and plasma insulin levels, indicating that Rapamycin
292
treatment improves metabolic parameters in db/db mice (Figure 1B). In addition, a
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dramatic reduction in lipid accumulation is observed in liver tissue of Rapamycin
294
treated animals (Figure 1C). To demonstrate target engagement of Rapamycin
295
treatment, phosphorylation of AMP activated protein Kinase (p-AMPK) and
296
phosphorylation of ribosomal protein S6 (p-prS6), a direct target of mTOR, were
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determined. Indeed, Rapamycin significantly increased p-AMPK expression and
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significantly reduced p-prS6 expression in liver tissue (Figure 1D).
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Diabetes accelerates experimental OA in db/db mice
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To investigate the influence of T2D in OA progression, Lean mice and db/db
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mice were subjected to experimental OA by transection of the medial meniscotibial
303
ligament and the medial collateral ligament in the right knee. Left knee joints were used
304
as an internal control. 10 weeks after surgery, right and left knee joints were evaluated
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ACCEPTED MANUSCRIPT by Safranin O staining. The results showed OA-like changes in the right knee in both
306
Lean and db/db mice (Figure 2 A,B). A significant increase in cartilage damage was
307
found in OA-db/db mice compared to OA-Lean mice (Figure 2C), while control knee
308
joints did not show histopathological cartilage changes. These results suggest first, that
309
diabetes accelerates cartilage damage after experimental OA and second, that this effect
310
is associated to metabolic disturbances of diabetic mice.
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mTOR inhibition prevents diabetes-accelerated experimental OA
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To evaluate if pharmacological mTOR inhibition has beneficial effects on
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diabetes-accelerated OA, db/db mice were subjected to experimental OA (N=16).
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Db/db-Vehicle mice exhibited OA-like changes with loss of proteoglycans and
317
structural changes in the surface lamina. On the other hand, db/db-Rapamycin mice only
318
showed a focal loss of proteoglycans and small structural changes in the articular
319
cartilage (Figure 2A,B), associated to a reduced histological OA score (Figure 2C).
320
These
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accelerated experimental OA in mice.
demonstrate
that
mTOR
inhibition
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Rapamycin reduces synovium inflammation in diabetes-accelerated experimental
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OA
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Inflammation is important in OA and T2D pathophysiology (33), although how
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bioenergy sensors link metabolism with inflammatory processes to regulate joint
327
integrity is unknown. We investigated how diabetes affects the synovium of db/db mice.
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In this sense, a significant increase of synovial inflammation is observed after surgery in
329
both Lean and db/db mice, although a greater inflammation score is found in db/db
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mice. Interestingly, Rapamycin treatment reduced synovial inflammation in db/db while
331
no signatures of histopathological changes were observed in the Control knee joints
332
(Figure 3A,B). These results suggest that the beneficial effect of mTOR inhibition in
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joints might be mediated by a reduction in synovium inflammation.
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Rapamycin reduces the expression of catabolic factors involved in cartilage loss
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and inflammation in db/db mice
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To evaluate the mechanism of action of diabetes-accelerated cartilage
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degradation in db/db mice, immunohistochemistry analysis was performed for MMP-
340
13, an important OA biomarker involved in cartilage degradation (34). The results
341
showed a significant increase in the expression of MMP-13 in OA db/db-Vehicle mice,
342
predominantly in the synovium area. Importantly, Rapamycin treated OA db/db mice
343
showed a significant reduction in the expression of MMP-13, indicating a potent anti-
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catabolic effect of mTOR inhibition in db/db mice (Figure 4A,B).
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To go further in the explanation of the protective effects of Rapamycin,
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proinflammatory cytokine levels were evaluated in plasma from db/db-Vehicle mice
347
and db/db-Rapamycin mice. The results showed a significant reduction in serum levels
348
of active interleukin 12 (IL-12p70), a proinflammatory cytokine (35), in db/db-
349
Rapamycin mice compared to db/db-Vehicle mice (p<0.01) (Figure 4C), suggesting a
350
potential common link between T2D and OA inflammatory mechanisms.
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Rapamycin maintains chondrocyte homeostasis in db/db mice
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Autophagy, a key homeostatic mechanism in articular cartilage, is reduced in
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chondrocytes from OA diabetic patients (11). To confirm this evidence, LC3, a main
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marker for autophagosome formation (36), was evaluated. The results showed that LC3
355
staining was reduced after experimental OA in db/db-Vehicle mice. Remarkably,
356
mTOR inhibition by Rapamycin maintains LC3 expression in db/db mice knee joints
357
subjected to experimental OA (Figure 5A). Chondrocyte cellularity in mouse knee joints was significantly reduced in
359
chondrocytes in OA-db/db-Vehicle mice compared to OA-Lean mice. Preservation of
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cellularity is observed in Rapamycin treated mice compared to OA db/db-Vehicle mice
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(Figure 5B).
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Together, these data suggest that cartilage homeostasis is compromised in db/db
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mice subjected to experimental OA, and that autophagy activation protects against
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chondrocyte loss.
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DISCUSSION Metabolism influences dramatically health outcomes, including articular
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cartilage and synovium function. However, the consequences of metabolic
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complications of diabetes in OA are not well understood. There are evidences indicating
384
that failure of homeostasis mechanisms such as autophagy, leads to alterations in
385
articular cartilage that ultimately cause joint damage and OA (20). Based on these
386
concepts, we previously demonstrated that high glucose and high insulin reduced
387
autophagy in human chondrocytes and accelerated cartilage degradation through
388
regulation of Akt/mTOR signaling pathway. We found that pharmacological activation
389
of autophagy attenuates the catabolic effects of high glucose and high insulin in human
390
chondrocytes. Our findings suggest that reduced autophagy might be one of the
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mechanisms responsible for the cartilage degradation observed in T2D patients (11).
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In the present study, we investigate the implication of T2D in OA development
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by using a genetic diabetes mice model. These mice have a mutation on the leptin
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receptor and lack leptin signaling, which leads to hyperphagia, persistent
395
hyperinsulinemia, hyperglycemia and obesity (37). Db/db mice exhibit metabolic
396
disturbances similar to those observed in humans with T2D, being a representive model
397
to study diabetes and the associated metabolic complications (38). Human metabolic
398
diseases have been linked to mTOR dysregulation (39), We evaluated how systemic
399
Rapamycin treatment, an autophagy inducer by mTORC1 inhibition, influences
400
metabolic phenotype of db/db mice. Rapamycin significantly improved body weight
401
gain, fasted blood glucose levels and serum insulin levels in db/db mice. To investigate
402
the link with the targets potentially responsible for these beneficial effects, we evaluated
403
the activity of AMPK, a key energy sensor that regulates cellular metabolism to
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405
established downstream activity, Rapamycin reduced p-rpS6 while increased the
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phosphorylation of AMPK. The data is a confirmation of the pharmacological activity
407
of Rapamycin in our model, related to AMPK response to energy stress probably
408
through inhibition of the mTORC1 pathway and autophagy activation (41).
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To study the connection between T2D and OA progression, we performed
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experimental OA in Lean and db/db mice. Our results showed that articular cartilage of
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OA-db/db mice developed more severe OA-like changes in articular cartilage compared
412
to OA-Lean mice. Notably, synovium inflammation and thickening was found in the
413
joints of OA-db/db mice. Joint inflammation was accompanied with an increase in
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MMP-13, predominantly in the synovial membrane. Together, these results support the
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concept that abnormal glucose metabolism, primarily associated with high levels of
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glucose and insulin accelerates experimental OA in db/db mice.
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The maintenance of autophagy is essential to preserve cartilage integrity.
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Decreased autophagy and increased chondrocyte death now established features of OA
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(42,43). In this sense, articular cartilage from diabetic mice displayed a reduction of
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LC3, and a decrease in cartilage cellularity, which suggest that joint damage observed in
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db/db mice might be due to defective autophagy which leads to an imbalance between
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the catabolic and anabolic pathways.
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Since decreased in autophagy could be one of the mechanisms responsible for
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the faster cartilage damage observed in diabetes, we evaluated whether pharmacological
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autophagy activation by Rapamycin could confer protection to joints. Indeed,
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Rapamycin treatment reduces the severity of experimental OA in C57BL/6J mice and
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prevents OA progression, consistent with previous observations (25-28). Our results
428
show that Rapamycin attenuates the severity of experimental OA in db/db mice,
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ACCEPTED MANUSCRIPT demonstrated by the diminished OA cartilage score in the treated mice. Moreover,
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Rapamycin treatment dramatically reduces synovial inflammation, the expression of
431
MMP-13 and circulating IL-12 levels were significantly reduced in response to
432
Rapamycin. Previous studies demonstrated an increase in IL-12 levels in serum from
433
patients with T2D (44) and in mice subjected to high fat diet and posttraumatic arthritis
434
(45), suggesting a potential common inflammatory pathway for diabetes and OA (46).
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In fact, OA cartilage from diabetic patients is more reactive to pro-inflammatory stress
436
than OA cartilage from non-diabetic patients, suggesting that diabetes increases
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inflammation in OA (47). Our results are consistent with previous findings where
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autophagy activation can prevent inflammatory responses (48,49).
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Furthermore, Rapamycin increases LC3 expression and maintains the
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chondrocyte number in db/db mice. These findings together with our previous
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observations suggest that Rapamycin treatment reduces joint damage in diabetic mice
442
with experimental OA by both normalizing altered metabolic parameters of T2D and by
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inducing autophagy.
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increased body weight and obesity, which is also an important risk factor for OA (37,
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45). Since we performed experimental OA only in right knee, left knee was employed as
447
a control to evaluate the direct effect of obesity in the joint in this time frame. We
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observed increased cartilage damage and synovial inflammation in the OA knee, while
449
control knee did not show any pathological changes. These results suggest that body
450
weight is not sufficient to accelerate experimental OA in db/db mice. One of the
451
limitations of this study is that contralateral limbs of DMM were used as control joints
452
as opposed to sham surgery joints. It is possible that an alteration of joint load can be
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developed as a result of experimental OA in the joint.
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To conclude, our study identifies a promising therapeutic approach to prevent
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cartilage degradation observed in T2D patients. However, Rapamycin, has some effects,
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which limits its use for preventive strategies in humans (50). Further studies are needed
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to identify new drugs that can activates autophagy and modify the OA progression in
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diabetic patients. This knowledge could leads to an improvement of clinical care in OA
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patients with metabolic diseases.
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ACCEPTED MANUSCRIPT Acknowledgements
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We thank the personal of the Experimental Unit for the animal maintenance.
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Purificación Filgueira and Noa Goyanes from the Histology Core and Lucia Lourido for
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technical assistance.
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Author contributions
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All authors approved the final version to be published.
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Dra. Caramés had full access to all of the data in the study and takes responsibility for
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the integrity of the data and the accuracy of the data analysis.
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Study conception and design. Mendes, Blanco, Caramés.
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Drafting and revising the manuscript. Ribeiro, López de Figueroa, Nogueira-Recalde,
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Centeno, Mendes, Blanco, Caramés.
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Acquisition of data. Ribeiro, López de Figueroa, Nogueira-Recalde, Caramés.
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Analysis and interpretation of data. Ribeiro, Caramés.
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Role of the Funding Source
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This study was supported by Instituto de Salud Carlos III-Ministerio de Economía y
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Competitividad, Spain-CP11/00095. Madalena Ribeiro was supported by Fundo Social
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Europeu (FSE), through "Programa Operacional Potencial Humano" (POPH),
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and national funds via FCT – Fundação Portuguesa para a Ciência e a Tecnologia under
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the PhD fellowship, SFRH/BD/78188/2011 and Beatriz Caramés was supported by
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Miguel Servet Program, Instituto de Salud Carlos III-CP11/00095.
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Competing interest
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The authors declare that there is no conflict of interest regarding the publication of this
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paper.
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Figure 1. Autophagy activation by Rapamycin improves metabolic parameters in
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db/db mice. A, Body weight of Lean, db/db-Vehicle and db/db-Rapamycin mice was
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evaluated every week during 10 weeks. Values are the means (ll, ul) 95% CI of eight
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mice in each experimental group. *p<0.0001 vs. Lean mice and **p (t0:p=0.4482,
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t1:p=0.2376, t2=0.0673, t3:p=0.0382, t4:p=0.0117, t5:p=0.0004, t6:p<0.0001, t7:p=0.0030,
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t8:p=0.0009, t9:p=0.0003, t10:p=0.0002) vs. db/db-Vehicle mice. B, Fasted blood
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glucose levels were determined after three measurements during the study. Values are
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the means (ll, ul) 95% CI of eight mice in each experimental group. Fasted plasma
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insulin levels were determined at the end of the study. Values are the means (ll, ul) 95%
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CI of eight mice in each experimental group. C, Liver sections from db/db-Vehicle and
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db/db-Rapamycin mice (N=8 each/group) were analyzed by staining with H&E.
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Representative images of liver sections from two mice per group. D, Liver protein
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extracts from db/db-Vehicle mice and db/db-Rapamycin mice. Phosphorylation of AMP
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activated protein kinase (AMPK) and ribosomal protein S6 (rpS6) was determined by
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Western blotting. Densitometric analysis of p-AMPK and p-rpS6. GAPDH and α-
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Tubulin were employed as a loading control. Values are the means (ll, ul) 95% CI of 5
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mice in each experimental group.
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Figure 2. Diabetes-accelerated experimental osteoarthritis is prevented by mTOR
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inhibition. Nine-week-old male db/db mice (B6.BKS (D)-Leprdb/J, N=16) and the
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respective background strain mice (C57BL/6J-non-diabetic lean mice, N=8) were
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subjected to experimental osteoarthritis (OA) in the right knee (OA). The left knee was
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not subjected to surgery and was used as a control (Ctrl). Three experimental mice
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groups were performed; Lean mice, db/db-Vehicle mice and db/db-Rapamycin mice.
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Each experimental group included eight mice. A-B, Knee joints were analyzed by
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histological scores of Lean, db/db-Vehicle and db/db-Rapamycin mice. Values are the
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means (ll, ul) 95% CI of eight mice in each experimental group (each point represents
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an individual mice).
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Figure 3. Effect of Rapamycin on synovial inflammation in experimental OA. A,
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Synovium from Lean, db/db-Vehicle and db/db-Rapamycin mice (Ctrl and OA, N=8
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each group) was analysed by staining with Hematoxylin Eosin (H&E). Scale bar
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100µm. Magnification: 10x. B, The histological score for inflammation was
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significantly increased in OA-db/db-Vehicle mice compared to OA-Lean mice and
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significantly decreased after Rapamycin treatment. Values are the means (ll, ul) 95% CI
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of eight mice in each experimental group (each point represents an individual mice).
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Figure 4. Rapamycin reduces the expression of catabolic factors implicated in the
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joint damage in db/db mice. A, Knee joints from Lean, db/db-Vehicle and db/db-
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Rapamycin mice were collected 10 weeks after surgery (N=5 each/group). Sections
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were analysed by immunohistochemistry for MMP-13. Two sections from one animal
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are presented. Scale bar 100µm. Magnification: 10x. B, Quantitative analysis of MMP-
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13 positive cells. Values are the means (ll, ul) 95% CI of five mice in each experimental
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group (each point represents the mean of two independent areas from each mice). C,
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Quantitative analysis of Interleukin-12 (IL12p70) in serum from db/db-Vehicle and
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db/db-Rapamycin mice. Values are the means (ll, ul) 95% CI of six mice in db/db-
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Vehicle group and eight mice in db/db-Rapamycin group (each point represents
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individual mice).
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Figure 5. Rapamycin maintains chondrocyte homeostasis in db/db mice. A, Knee
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joint from Lean, db/db-Vehicle and db/db-Rapamycin mice with experimental OA (N=3
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autophagosome formation and autophagy activation. Scale bar 100µm. Magnification:
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10x. B, Quantitative analysis of cell number in OA-Lean, OA-db/db-Vehicle and OA-
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db/db-Rapamycin mice (N=5 each/group). Values are the means (ll, ul) 95% CI of five
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mice in each experimental group (each point represents the mean of three independent
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areas for each mice).
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