- Email: [email protected]
Rosiglitazone reduces body wasting and improves survival in a rat model of cancer cachexia
Accepted Manuscript Rosiglitazone reduces body wasting and improves survival in a rat model of cancer cachexia Katja Trobec, Sandra Palus, Anika Tschirner, Stephan von Haehling, MD, PhD Wolfram Doehner, MD, PhD Mitja Lainscak, MD, PhD Stefan D. Anker, MD, PhD Jochen Springer, PhD PII:
S0899-9007(13)00556-X
DOI:
10.1016/j.nut.2013.12.005
Reference:
NUT 9181
To appear in:
Nutrition
Received Date: 12 October 2013 Revised Date:
5 December 2013
Accepted Date: 5 December 2013
Please cite this article as: Trobec K, Palus S, Tschirner A, von Haehling S, Doehner W, Lainscak M, Anker SD, Springer J, Rosiglitazone reduces body wasting and improves survival in a rat model of cancer cachexia, Nutrition (2014), doi: 10.1016/j.nut.2013.12.005. 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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Title: Rosiglitazone reduces body wasting and improves survival in a rat model of cancer cachexia
Authors: Katja Trobec Haehling, MD, PhD
2,3
1*
, Sandra Palus
2,3*
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Running head: Rosiglitazone improves survival in cancer cachexia
, Anika Tschirner
, Wolfram Doehner, MD, PhD
2,4
2,3
, Stephan von
, Mitja Lainscak, MD, PhD
,
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Stefan D. Anker, MD, PhD 6, Jochen Springer, PhD 2,3,7
2, 5
Affiliations:
Pharmacy department, University Clinic Golnik, Golnik, Slovenia
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Applied Cachexia Research, Department of Cardiology, Charité Medical School,
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Berlin, Germany
Center for Cardiovascular Research, Charité Medical School, Berlin, Germany
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Center for Stroke Research Berlin, Charité Medical School, Berlin, Germany
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Division of Cardiology, University Clinic Golnik, Golnik, Slovenia
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Centre for Clinical and Basic Research, IRCCS San Raffaele, Rome, Italy
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Norwich Medical School, University of East Anglia, Norwich, UK
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: equal contribution
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Role of each author:
Study design: JS, SP, SDA, WD In-vivo work: SP, AT, JS In-vitro work: AT, KT Statistical analysis: JS, SvH Manuscript preparation: KT, ML, SDA, JS
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Word count: 4399 words (abstract: 250, text: 3811, tables: 338) Number of figures: 4
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Number of tables: 2
Corresponding author:
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Jochen Springer Center for Cardiovascular Research
Hessische Str. 3-4, 10115 Berlin, Germany Tel: +49-30-450-525023 Fax: +49-30-450-525978
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E-mail: [email protected]
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ABSTRACT
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Objective
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Rosiglitazone improves insulin sensitivity and promotes weight gain in patients with
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Diabetes Mellitus type 2, which could be useful in wasting and cachexia. However, its
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effects on cardiac function are controversial. We aimed to investigate the effects of
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rosiglitazone on body wasting, body composition, cardiac function and survival in a
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rat model of cancer cachexia.
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Methods
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Rats were injected Yoshida AH-130 hepatoma tumor cells and randomized to receive
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placebo or rosiglitazone 4 mg/kg/d. Treatment started one day after tumor inoculation
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and the rats were sacrificed 14 days thereafter. Body weight and body composition
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was measured at baseline and after the removal of the tumor. Echocardiography was
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performed at baseline and on day 11. At the end of the study, organs were weighed
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and the proteasome activity in gastrocnemius muscle was measured.
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Results
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Survival analysis showed a significant benefit from treatment with rosiglitazone
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(hazard ratio = 0.38, 95% confidence interval: 0.15 – 0.86). Rosiglitazone reduced
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average daily weight loss (2.33 g/d rosiglitazone vs. 3.93 g/d placebo, p < 0.05) as a
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result of both fat and lean mass preservation. It decelerated white and brown tissue
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wasting, but had no effect on skeletal muscle mass and heart mass. However,
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peptidyl-glutamyl-protein-hydrolysing and trypsin-like activity in gastrocnemius
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muscle was significantly reduced by rosiglitazone. Finally, it increased left ventricular
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ejection fraction, fractional shortening and systolic volume and improved cardiac
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output in cachectic cancer rats.
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Conclusions
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Rosiglitazone prevents weight loss and improves survival in rat model of cancer
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cachexia. It exerts beneficial effects on cardiac function.
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composition, echocardiography
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Key Words: glitazone, thiazolidinedione, wasting, proteasome, survival, body
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1. Introduction
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Wasting is common in patients with cancer and ranges from 31% in patients with
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sarcoma to nearly 90% in pancreatic cancer [1, 2]. Weight loss can lead to cachexia
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with further aggravation of symptomatic status and overall decline of exercise
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capacity. Finally, tissue wasting and the development of cachexia may ensue, both
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being a powerful independent marker of impaired survival [1, 3].
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In cancer cachexia, various metabolic pathways are abnormally regulated to cause
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catabolic/anabolic imbalance. One of the main mechanisms suggested to be involved
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in development of cachexia and muscle wasting, is insulin resistance (IR) [4, 5]. IR is
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responsible for activation of ubiquitin proteasome system (UPS) in muscle, which
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involves up-regulation of Atrogin-1/MAFbx and muscle RING-finger protein-1 (MuRF-
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1) [6], and is considered to be the main mechanism of muscle wasting in humans [7].
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The thiazolidinediones (glitazones, TZDs) are selective ligands of the nuclear
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transcription factor peroxisome-proliferator-activated receptor γ (PPARγ) that exert
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specific metabolic action towards increasing insulin sensitivity [8]. They are widely
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used in the treatment of diabetes mellitus (DM) type 2, but may cause weight gain,
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which is regarded as unwanted process in this population [9]. However, this might be
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a favourable effect in patients with cachexia, especially since insulin sensitizers were
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shown to attenuate lean mass loss in older patients with DM [10]. In a mouse model
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of cancer cachexia, rosiglitazone (RSG) was shown to reduce weight loss [11, 12].
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It has to be considered, however, that the effects of TZDs on heart function are
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somewhat controversial. Therapy with TZDs results in increased fluid retention [13], 5
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which has led the authorities to discourage the use of TZDs in heart failure patients
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[14]. Alternatively, echocardiographic studies showed that TZDs after 52 weeks of
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therapy do not negatively affect myocardial structure or function [15]. RSG was
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shown to induce eccentric cardiac hypertrophy, associated with an increase in
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myofibrillar protein content and turnover and slightly increased stroke volume in
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healthy rats [16], which could be favorable in cancer cachexia, where cardiac
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remodeling and atrophy impair cardiac function [17].
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We aimed to investigate the effects of rosiglitazone (RSG) on body wasting, body
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composition and survival in a rat Yoshida AH-130 hepatoma cancer cachexia model.
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Due to safety concerns, we also aimed to assess the effect of RSG on cardiac
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function.
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2. Materials and methods
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2.1. Tumour Model
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Male Wister Han rats (mean weight 201.6 ± 1.6 g, age 7-8 weeks) were injected
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intra-peritoneally with 108 Yoshida AH-130 tumour cells as described previously [18].
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2.2. Study design
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After tumor inoculation rats were randomized into the placebo (n = 49) or RSG 4
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mg/kg/day (n = 15) group. The control group (n = 10) was injected with saline. Before
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tumour inoculation, baseline weight, body composition and quality of life indicators
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were measured (Figure 1). Treatment with placebo (water) or active compound,
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given per gavage, started one day after tumour inoculation. All staff handling the rats
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was blinded to treatment allocation. Animals were housed under specific pathogen
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free
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cardiac function was analysed and quality of life was assessed once more. Animals
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were euthanized 14 days after tumour inoculation. Several animals had to be
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euthanized prematurely for ethical reasons according to prospectively defined criteria
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of disease burden (Figure 1) [19]. Body weight and body composition were assessed
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every second day, but the final assessment of body weight and body composition
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was performed after removal of the tumour. At the end of the study, organs were
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removed and weighed. All procedures were approved by the local animal ethics
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committee.
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conditions in groups of three and were monitored twice daily. On day 11,
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2.3. Body composition
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Body composition (fat and lean body mass) was analysed with Nuclear Magnetic
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Resonance spectroscopy (EchoMRI-700, Echo Medical Systems, Houston, TX), as
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previously described [18].
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2.4. Echocardiography
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Echocardiography was performed as described before [20]. Briefly, rats were
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anesthetized using 1.5% isoflurane and laid in supine position on a platform. Body
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temperature was monitored and maintained at 36–38°C using a heating pad. All hair
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was removed from the chest using a chemical hair remover. B- and M-mode tracings
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were recorded and assessed for left ventricular dimensions and function using the
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high resolution Vevo 770 system (Visual Sonics, Toronto, Canada).
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2.5. Quality of life indicators
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Quality of life in animals can be assessed by measurement of spontaneous activity
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and food intake [21]. Rats were housed individually over a period of 24 hours. Food
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intake was recorded and spontaneous movement was assessed by an infrared
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monitoring system (Supermex, Muromachi, Tokyo, Japan).
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2.6. Proteasome activity
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At the end of the study, the proteolytic activity of the proteasome was analysed as
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described before [19]. Briefly, protein isolated from gastrocnemius muscle were
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incubated with one of the fluorogenic substrates (Biomol, Hamburg, Germany) to
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determine
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methylcoumarin [Z-LLE-AMC]), chymotrypsin-like activity (succinyl-Leu-Leu-Val-Tyr-
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7-amido-4-methylcoumarin [LLVY-AMC]) or peptidyl-glutamyl-protein-hydrolysing
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trypsin-like
activity
(benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-
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activity (benzoyl- Val-Gly-Arg-7-amidocoumarin [Bz-VGR-AMC]), at 37°C for 1 hour
2
followed by 10 minutes on ice. The fluorescence intensity was measured with a
3
fluorometer (Twinkle LB 970, Berthold, Bad Wildbad, Germany) at 360nm excitation
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and 460nm emission. The activity, expressed as nmol/mg protein/min, was
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calculated by using free amidomethylcoumarin (AMC) as working standard.
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2.7. Statistics
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Data were analyzed using GraphPad PRISM 5.0 (GraphPad Software, Inc, La Jolla,
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CA, USA). Results are shown as mean ± standard error of the mean (SEM). All data
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were tested for normal distribution using the D'Agostino & Pearson omnibus
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normality test. Between-group comparison was performed using ANOVA followed by
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Tukey’s tests for data with normal distribution; data with skewed distribution were
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analysed by Kruskal-Wallace and Dunns test. All statistical tests were two sided and
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a p-value < 0.05 was considered significant. Survival was tested by Cox proportional
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hazard analysis. Hazard ratio (HR) and 95% confidence interval (CI) are shown. A p
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value of < 0.05 was considered significant.
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3. Results
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3.1. Survival
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Treatment with 4 mg/kg/d RSG had no effect on tumour growth (6.3 ± 0.7 and 5.8 ±
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0.5 x 109 cells for RSG and placebo, respectively). The survival analysis shows a
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significant benefit from treatment with RSG in AH-130 tumor bearing rats, with a risk
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reduction of 62% (HR = 0.38, 95% CI: 0.15 – 0.86, p < 0.05; Figure 2). Survival rate
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at day 14 was 80% in RSG group compared to 48% in placebo group.
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3.2. Body mass and body composition
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Tumor bearing animals lost weight, but this loss was reduced with RSG treatment
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(Figure 3). The average loss of body mass per day was 3.93 ± 0.2 g in placebo
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animals compared to 2.33 ± 0.81 g in RSG treated animals (p < 0.05). Reduction of
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body mass resulted from both fat and lean mass wasting. The average loss of lean
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mass per day was 2.93 ± 0.15 g in the placebo group and 2.00 ± 0.55 g in the treated
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group (p < 0.001). The average loss of fat mass per day was 0.95 ± 0.04 g in the
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placebo group and 0.49 ± 0.18 g in the treated group (p < 0.001).
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3.3. Organ weight
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Mass of all organs was significantly lower in cachectic cancer rats (placebo group)
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when compared to controls (p < 0.001, Table 1). Liver mass, heart mass and mass of
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skeletal muscles were not significantly different in RSG group when compared to
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placebo. The mass of white adipose tissue (WAT), brown adipose tissue (BAT) and
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spleen was significantly higher in RSG group (p < 0.05 versus placebo).
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3.4. Cardiac function
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Cardiac function was similar in both groups before tumour inoculation (data not
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shown), but was improved by treatment with RSG compared to placebo on day 11
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(Table 2). There was no difference in left ventricular mass, in the diameters of the left
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ventricle and volumes as well as thickness of posterior wall, both in diastole and
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systole. However, there was a greater thickness of the septal wall in systole and an
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improved fractional shortening (41.2% in RSG group vs. 30.5% in placebo group, p <
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0.01), ejection fraction (62.4% in RSG group vs. 52.5% in placebo group, p < 0.05)
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and stroke volume (147.3 µL in RSG group vs. 106.8 µL in placebo group, p < 0.01).
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The result of the improved pumping function, i.e. systolic function, was a significantly
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higher cardiac output. The diastolic function assessed as the ratio of E-and A-wave
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of the mitral blood flow was within the normal range in both groups and not
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statistically different.
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3.5. Quality of life
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Food intake and spontaneous locomotor activity were not different before inoculation
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of the tumor cells. On day 11 there was a significant improvement of food intake by
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RSG (126%) compared to placebo (7.93 ± 2.09 vs. 4.30 ± 0.48 g/24h, respectively, p
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= 0.01). Locomotor activity was also higher in RSG (41%) treated animals 41,543 ±
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5,327 vs. 29,509 ± 1,775 beam breaks/24h, p < 0.05).
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3.6. Proteasome activity
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Peptidyl-glutamyl-protein-hydrolysing, trypsin-like and chymotrypsin-like activities
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were all increased in the gastrocnemius muscle of tumor-bearing animals. RSG
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significantly reduced the peptidyl-glutamyl-protein-hydrolysing activity (319 ± 28
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nmol/mg protein/min vs. 167 ± 62 nmol/mg protein/min, p < 0.05) and trypsin-like
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activity (179 ± 33 nmol/mg protein/min vs. 88 ± 40 nmol/mg protein/min, p < 0.05) in
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cachectic rats compared to placebo. RSG did not affect the chymotrypsin-like activity
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(Figure 4).
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4. Discussion
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We have shown for the first time that RSG improves survival and quality of life in rats
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with cancer cachexia along with having favorable effects on cardiac function.
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Improved survival of rats is probably the result of weight loss prevention, since weight
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loss is long known to be associated with impaired survival in cancer patients [1].
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RSG was shown before to reduce the weight loss in tumor-bearing mice. However, it
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was effective in preventing the weight loss in early cachexia, but had no effect in
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slowing the rate of weight loss in late-stage cachexia. [11, 12]. Time dependent
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manner of body weight and adipose tissue loss in rats with cancer cachexia was also
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shown by Batista et al., who observed significant loss no earlier than day 14 after
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tumor inoculation [22]. In our study, weight was measured only at baseline and at the
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end of the study, so we could not observe the time course of weight loss. However,
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the mechanisms of weight loss may have changed trough the course of the disease.
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With regard to body composition, RSG decelerated the loss of both fat and lean
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mass. In cancer cachexia, fat tissue precedes lean tissue loss and is related to
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reduction of adipocyte size, not number. Lipolysis is increased and so is the turnover
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of glycerol and free-fatty acids; however, the exact mechanism of fat tissue loss
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remains unknown [23, 24]. PPARγ expression, which is essential for adipocyte
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differentiation, proliferation, fatty acid uptake and storage, is reduced in WAT [22].
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TZDs induce WAT lipolysis but also increase the uptake of free-fatty acids by WAT
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and improve the response of WAT lipolysis to the inhibitory action of insulin [25]. In
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DM type 2 patients, RGS was shown to contribute to redistribution of regional body
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fat [26, 27]. In our study, RSG prevented the loss of fat mass in cancer cachexia,
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which was confirmed with both body composition determination and measurement of
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WAT and BAT mass at the end of the study. Same was observed in the recently
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published studies of Asp et al. [11, 12].
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Body composition measurement also revealed the beneficial effect of RSG on lean
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mass wasting; however, the organ weights of skeletal muscle, heart and liver – while
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higher - were not significantly different in RSG group compared to placebo. A similar
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lack of effect was observed in studies of Asp et al. [11, 12]. In skeletal muscle,
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cancer cachexia increases the expression of Atrogin-1 and MuRF-1, which leads to
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increased protein degradation. Concomitantly, myofibrillar protein synthesis is
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reduced [28]. Although we did not show beneficial effect of RSG on skeletal muscle
18
mass, RSG reduced the activity of proteasome system in gastrocnemius muscle.
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RSG evidently decelerates muscle protein degradation, which could be the
20
consequence of IR improvement. In the study of Wang et al., IR was shown to
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accelerate muscle protein degradation and RSG was able to reduce it by lowering
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the expression of Atrogin-1/MAFbx and MuRF1. However, only partial recovery of the
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cross-sectional area and muscle mass was achieved. It was suggested that the
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recovery was not complete because RSG did not stimulate protein synthesis [7]. The
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effect of RSG may also change trough the progression of cachexia. Asp et al.
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showed that RSG reduced gene expression of Atrogin-1 and MuRF-1 in early
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cachexia but not in late-stage cachexia [12].
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Besides tissue wasting in cancer cachexia, heart function also deteriorates. In our
5
study, rats with cancer had reduced left ventricular ejection fraction, fractional
6
shortening, stroke volume, cardiac output, and other abnormalities. Reduced cardiac
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contractile function in cancer cachexia results from cardiac remodeling and atrophy.
8
Fibrosis, disrupted alignments of sarcomeric structure and impaired integrity of
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mitochondria can be seen in cardiac muscle cells [29]. Cardiomyocyte function is
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depressed during both cellular contraction and relaxation [30].On molecular level,
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myocardium protein degradation is increased with rise in expression of MuRF-1 and
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Atrogin-1 and increased level of protein ubiquitination. Additionally, the expression of
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glucose transporter type 4 (GLUT4) is decreased [29].
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In our study, RSG improved cardiac function in rats with cancer cachexia. It
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increased left ventricular ejection fraction (LVEF) and cardiac output in comparison to
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rats receiving placebo. Festuccia et al. have shown that in healthy rats, RSG induces
18
eccentric cardiac hypertrophy, reduces heart rate and slightly increases stroke
19
volume. It switches from fatty acids to glucose as a major source of energy substrate
20
and causes increased turnover of myofibrillar proteins: it increases synthesis,
21
enhances calpain-mediated myofibrillar degradation, but reduces protein degradation
22
through the UPS [25]. In our study, however, a hypertrophy of the heart muscle was
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not detected - the heart mass and mass of left ventricle were not significantly higher
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in the RSG group when compared to placebo.
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In vitro studies showed that activation of PPARγ receptors inhibits malignant cell
2
proliferation and suppresses angiogenic tumor phenotype [31, 32]. In rats, TZD were
3
shown to suppress carcinogenesis [33, 34], although the results are somewhat
4
contradictive [35]. Additionally, recent meta-analysis of clinical data showed reduced
5
risk of liver cancer in DM type 2 patients receiving TZD [36]. In our study, however,
6
RSG did not affect the tumor growth, since the number of the tumor cells did not
7
differ significantly between placebo and RSG group.
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In the light of translating the findings of our study to human population, one must
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consider the increased risk of cardiovascular events in patients, treated with RSG
11
that has already led to withdrawal of the drug from the market. In order make the
12
drug applicable to cancer patients, the benefits of attenuated weight loss should
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outweigh the increased risk for cardiovascular events. Further studies are needed to
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address this issue.
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5. Limitations
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The body weight and body composition of the rats were recorded every two days;
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however, due to the tumor growth, we were not able to determine if weight loss and
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changes in body composition occurred in a time dependent manner. Measurement
20
of insulin sensitivity could have provided more detailed insight in the molecular
21
mechanisms of RSG effects.
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6. Conclusion
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In conclusion, RSG improves survival, quality of life and decelerates weight loss in
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the rat model of cancer cachexia, which could be useful in cancer patients. Despite
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discouragement of TZD use in heart failure, RSG could have beneficial effects in
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cancer patients, where heart function is worsened by cancer cachexia.
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rosiglitazone on echocardiographic function and cardiac status in type 2
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diabetic patients with New York Heart Association Functional Class I or II Heart Failure. J Am Coll Cardiol 2007;49(16):1696-704.
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16. Festuccia WT, Laplante M, Brûlé S, Houde VP, Achouba A, Lachance D, et al.
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Rosiglitazone-induced heart remodelling is associated with enhanced turnover
19
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of myofibrillar protein and mTOR activation. J Mol Cell Cardiol 2009;47(1):85-
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17. Tian M, Nishijima Y, Asp ML, Stout MB, Reiser PJ, Belury MA. Cardiac
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alterations in cancer-induced cachexia in mice. Int J Oncol 2010;37(2):347-53.
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18. Schmidt K, von Haehling S, Doehner W, Palus S, Anker SD, Springer J. IGF-1
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Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat
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model of cancer cachexia. Int J Cancer 2012;131(9):2187-96.
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20. Akashi YJ, Palus S, Datta R, Halem H, Taylor JE, Thoene-Reineke C, et al.
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No effects of human ghrelin on cardiac function despite profound effects on
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21. Bauhofer A, Witte K, Celik I, Pummer S, Lemmer B, Lorenz W. Sickness behaviour, an animal equivalent to human quality of life, is improved in septic rats by G-CSF and antibiotic prophylaxis. Langenbecks Arch Surg 2001;386(2):132-40.
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22. Batista ML Jr, Neves RX, Peres SB, Yamashita AS, Shida CS, Farmer SR, et
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al. Heterogeneous time-dependent response of adipose tissue during the
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development of cancer cachexia. J Endocrinol 2012;215(3):363-73.
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23. Byerley LO, Lee SH, Redmann S, Culberson C, Clemens M, Lively MO.
Evidence for a novel serum factor distinct from zinc alpha-2 glycoprotein that
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promotes body fat loss early in the development of cachexia. Nutr Cancer
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2010;62(4):484-94.
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24. Rydén M, Arner P. Fat loss in cachexia--is there a role for adipocyte lipolysis? Clin Nutr 2007;26(1):1-6.
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25. Festuccia WT, Laplante M, Berthiaume M, Gélinas Y, Deshaies Y. PPARgamma agonism increases rat adipose tissue lipolysis, expression of
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glyceride lipases, and the response of lipolysis to hormonal control.
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Diabetologia 2006;49(10):2427-36.
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26. Virtanen KA, Hällsten K, Parkkola R, Janatuinen T, Lönnqvist F, Viljanen T, et
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al. Differential effects of rosiglitazone and metformin on adipose tissue distribution and glucose uptake in type 2 diabetic subjects. Diabetes 2003;52(2):283-90.
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27. Nam JS, Nam JY, Yoo JS, Cho M, Park JS, Ahn CW, et al. The effect of
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rosiglitazone on insulin sensitivity and mid-thigh low-density muscle in patients
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with Type 2 diabetes. Diabet Med 2010;27(1):30-6. 21
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28. White JP, Baynes JW, Welle SL, Kostek MC, Matesic LE, Sato S, et al. The regulation of skeletal muscle protein turnover during the progression of cancer
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cachexia in the Apc(Min/+) mouse. PLoS One 2011;6(9):e24650.
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29. Tian M, Asp ML, Nishijima Y, Belury MA. Evidence for cardiac atrophic
remodeling in cancer-induced cachexia in mice. Int J Oncol 2011;39(5):1321-
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6.
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7
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30. Xu H, Crawford D, Hutchinson KR, Youtz DJ, Lucchesi PA, Velten M, et al.
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Myocardial dysfunction in an animal model of cancer cachexia. Life Sci
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2011;88(9-10):406-10.
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15
31. Hatton JL, Yee LD. Clinical use of PPARgamma ligands in cancer. PPAR Res
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2008;2008:159415.
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32. Panigrahy D, Shen LQ, Kieran MW, Kaipainen A. Therapeutic potential of
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thiazolidinediones as anticancer agents. Expert Opin Investig Drugs
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2003;12(12):1925-37.
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33. Borbath I, Leclercq I, Moulin P, Sempoux C, Horsmans Y. The PPARgamma
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agonist pioglitazone inhibits early neoplastic occurrence in the rat liver. Eur J
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Cancer 2007;43(11):1755-63.
25
22
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34. Shen B, Chu ES, Zhao G, Man K, Wu CW, et al. PPARgamma inhibits
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hepatocellular carcinoma metastases in vitro and in mice.Br J Cancer
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2012;106(9):1486-94.
5
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35. Lubet RA, Fischer SM, Steele VE, Juliana MM, Desmond R, Grubbs CJ. Rosiglitazone, a PPAR gamma agonist: potent promoter of
7
hydroxybutyl(butyl)nitrosamine-induced urinary bladder cancers. Int J Cancer
8
2008;123(10):2254-9.
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10
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36. Wang F, Zhao SZ, Zhang MY, Ma YL, Zhang P, Qin HL. Decreased risk of liver cancer with thiazolidinediones therapy in patients with type 2 diabetes:
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results from a meta-analysis. Hepatology 2013 Jan 16. doi:
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10.1002/hep.26259. [Epub ahead of print]
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37.
2
FIGURE LEGENDS
3
Figure 1. Flow chart of experimental protocol.
4
D = day, QoL = quality of life, Echo = echocardiography
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Figure 2. Survival analysis. RSD improves survival in tumor-bearing rats.
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Figure 3. Changes in body weight and body composition. There were no differences
9
between the groups at baseline. The profound loss of body weight of bearing rats
10
was attenuated by RSG. A similar effect was seen for lean mass and fat mass. * p <
11
0.05, ** p < 0.01, *** p < 0.001: baseline versus day 14; # p < 0.05, ## p < 0.01, ###
12
p < 0.001: control/RSG versus placebo on day 14
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Figure 4. Proteasome activity. RSG reduces peptidyl-glutamyl-protein-hydrolysing
15
(PGPH) and trypsin-like activity, but not chymotrypsin-like activity of the proteasome.
16
* p < 0.05: RSG/control versus placebo
17
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Table 1: Organ weight Placebo
Rosiglitazone
(n = 10)
(n = 49)
4mg/kg/d (n = 15)
liver [mg]
10090±300***
6228±186
6723±481
spleen [mg]
633±26***
187±12
WAT [mg]
1348±90***
93±26
332±173*
BAT [mg]
279±23***
89±4
123±22*
GC [mg]
1179±30***
725±17
tibialis [mg]
428±7***
265±6
soleus [mg]
94±3***
EDL [mg]
100±2***
heart [mg]
751±8***
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Control
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252±40*
794±55 289±17
70±1
66±4
64±1
67±4
506±12
526±26
WAT = white adipose tissue, BAT = brown adipose tissue, GC = gastrocnemius, EDL = extensor
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digitalis longus
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* p < 0.05, *** p < 0.001 versus placebo
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Table 2: Cardiac function Placebo
Rosiglitazone
(n = 10)
(n = 40)
4 mg/kg/d (n = 15)
LVEF [%]
67.8 ± 2.5*
52.5 ± 2.0
62.4 ± 2.0*
LVFS [%]
50.4 ± 2.1**
30.5 ± 1.7
LVEDD [mm]
6.37 ± 0.11*
5.74 ± 0.12
LVESD [mm]
3.44 ± 0.19*
3.98 ± 0.11
LVEDV [µL]
269.4 ± 14.5**
199.9 ± 9.8
236.0 ± 16.7
LVESV [µL]
85.5 ± 6.0
93.1 ± 4.9
88.7 ± 7.5
LVSV [µL]
183.9 ± 13.4***
Heart rate [bpm]
413 ± 10
CO [mL/min]
78.85 ± 6.57***
LVPW dia [mm]
1.86 ± 0.13
LVPW sys [mm]
2.96 ± 0.15***
Septum dia [mm]
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Control
41.2 ± 2.1** 6.16 ± 0.17
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3.61 ± 0.15
147.3 ± 11.8**
375 ± 13
371 ± 9
41.97 ± 2.97
56.38 ± 5.74*
1.68 ± 0.05
1.53 ± 0.06
2.18 ± 0.06
2.22 ± 0.07
1.56 ± 0.07
1.47 ± 0.04
1.32 ± 0.04*
Septum sys [mm]
2.82 ± 0.09***
2.15 ± 0.05
2.40 ± 0.07*
LV mass [mg]
574 ± 42***
430.5 ± 11.3
420.2 ± 19.2
1.57 ± 0.19
1.36 ± 0.09
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106.8 ± 6.8
1.57 ± 0.10
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LV = left ventricular, EF = ejection fraction, FS = fractional shortening, EDD = end-diastolic diameter, ESD = end-systolic diameter, EDV = end-diastolic volume, ESV = end-systolic volume, SV = stroke volume, CO = cardiac output, PW = posterior wall, dia = diastole, sys = systole * p < 0.05, ** p < 0.01, *** p < 0.001 versus placebo
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Sacrificed animals
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n=3
n = 15
D11
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Tumor inoculation
D0
Placebo group
n = 26
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n = 49
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Body weight Body composition Echo QoL
n = 12
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RSG group
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STUDY END D14
D1
n = 23 Sacrificed animals
D11
D14
Echo QoL
Body weight Body composition Organ weight
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Survival proportions [%]
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100 80 60
20 0 4
6
8
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10
12
days after tumor inoculation
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14
4m g/kg/d rosiglitazone (n=15) placebo (n=49)
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body weight [g]
80 60
300 ###
40
***
***
###
**
20 #
0 -20
100
-40 -60
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200
delta body weight [g]
control
0 14 control
0 14 0 14 placebo rosiglitazone
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0
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lean mass [g] 250
rosiglitazone
delta lean mass [g]
60
**
***
***
###
40
###
200 150
placebo
###
20
#
0
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100 50 0
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30 ###
***
***
#
-60
0 14 0 14 placebo rosiglitazone
fat mass [g]
20
-40 control
placebo
rosiglitazone
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0 14 control
-20
delta fat mass [g] 15 ###
10 5
* #
10
0 -5 -10
0
0 14 control
0 14 0 14 placebo rosiglitazone
###
-15 control
placebo
rosiglitazone
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PGPH activity
300
* 200
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nmol/mg protein/min
400
100 0 placebo
rosiglitazone
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control
200 150
*
*
100
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nmol/mg protein/min
250
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trypsin-like activity
50 0
placebo
rosiglitazone
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control
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chymotrypsin-like activity
nmol/mg protein/min
30
20
10
0 control
placebo
rosiglitazone
S0899-9007(13)00556-X
DOI:
10.1016/j.nut.2013.12.005
Reference:
NUT 9181
To appear in:
Nutrition
Received Date: 12 October 2013 Revised Date:
5 December 2013
Accepted Date: 5 December 2013
Please cite this article as: Trobec K, Palus S, Tschirner A, von Haehling S, Doehner W, Lainscak M, Anker SD, Springer J, Rosiglitazone reduces body wasting and improves survival in a rat model of cancer cachexia, Nutrition (2014), doi: 10.1016/j.nut.2013.12.005. 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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Title: Rosiglitazone reduces body wasting and improves survival in a rat model of cancer cachexia
Authors: Katja Trobec Haehling, MD, PhD
2,3
1*
, Sandra Palus
2,3*
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Running head: Rosiglitazone improves survival in cancer cachexia
, Anika Tschirner
, Wolfram Doehner, MD, PhD
2,4
2,3
, Stephan von
, Mitja Lainscak, MD, PhD
,
SC
Stefan D. Anker, MD, PhD 6, Jochen Springer, PhD 2,3,7
2, 5
Affiliations:
Pharmacy department, University Clinic Golnik, Golnik, Slovenia
2
Applied Cachexia Research, Department of Cardiology, Charité Medical School,
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Berlin, Germany
Center for Cardiovascular Research, Charité Medical School, Berlin, Germany
4
Center for Stroke Research Berlin, Charité Medical School, Berlin, Germany
5
Division of Cardiology, University Clinic Golnik, Golnik, Slovenia
6
Centre for Clinical and Basic Research, IRCCS San Raffaele, Rome, Italy
7
Norwich Medical School, University of East Anglia, Norwich, UK
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: equal contribution
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*
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Role of each author:
Study design: JS, SP, SDA, WD In-vivo work: SP, AT, JS In-vitro work: AT, KT Statistical analysis: JS, SvH Manuscript preparation: KT, ML, SDA, JS
1
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Word count: 4399 words (abstract: 250, text: 3811, tables: 338) Number of figures: 4
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Number of tables: 2
Corresponding author:
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Jochen Springer Center for Cardiovascular Research
Hessische Str. 3-4, 10115 Berlin, Germany Tel: +49-30-450-525023 Fax: +49-30-450-525978
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E-mail: [email protected]
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Charité, Campus Mitte
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ABSTRACT
2
Objective
4
Rosiglitazone improves insulin sensitivity and promotes weight gain in patients with
5
Diabetes Mellitus type 2, which could be useful in wasting and cachexia. However, its
6
effects on cardiac function are controversial. We aimed to investigate the effects of
7
rosiglitazone on body wasting, body composition, cardiac function and survival in a
8
rat model of cancer cachexia.
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Methods
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Rats were injected Yoshida AH-130 hepatoma tumor cells and randomized to receive
12
placebo or rosiglitazone 4 mg/kg/d. Treatment started one day after tumor inoculation
13
and the rats were sacrificed 14 days thereafter. Body weight and body composition
14
was measured at baseline and after the removal of the tumor. Echocardiography was
15
performed at baseline and on day 11. At the end of the study, organs were weighed
16
and the proteasome activity in gastrocnemius muscle was measured.
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Results
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Survival analysis showed a significant benefit from treatment with rosiglitazone
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(hazard ratio = 0.38, 95% confidence interval: 0.15 – 0.86). Rosiglitazone reduced
21
average daily weight loss (2.33 g/d rosiglitazone vs. 3.93 g/d placebo, p < 0.05) as a
22
result of both fat and lean mass preservation. It decelerated white and brown tissue
23
wasting, but had no effect on skeletal muscle mass and heart mass. However,
24
peptidyl-glutamyl-protein-hydrolysing and trypsin-like activity in gastrocnemius
25
muscle was significantly reduced by rosiglitazone. Finally, it increased left ventricular
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ejection fraction, fractional shortening and systolic volume and improved cardiac
2
output in cachectic cancer rats.
3
Conclusions
5
Rosiglitazone prevents weight loss and improves survival in rat model of cancer
6
cachexia. It exerts beneficial effects on cardiac function.
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composition, echocardiography
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Key Words: glitazone, thiazolidinedione, wasting, proteasome, survival, body
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1. Introduction
3
Wasting is common in patients with cancer and ranges from 31% in patients with
4
sarcoma to nearly 90% in pancreatic cancer [1, 2]. Weight loss can lead to cachexia
5
with further aggravation of symptomatic status and overall decline of exercise
6
capacity. Finally, tissue wasting and the development of cachexia may ensue, both
7
being a powerful independent marker of impaired survival [1, 3].
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In cancer cachexia, various metabolic pathways are abnormally regulated to cause
10
catabolic/anabolic imbalance. One of the main mechanisms suggested to be involved
11
in development of cachexia and muscle wasting, is insulin resistance (IR) [4, 5]. IR is
12
responsible for activation of ubiquitin proteasome system (UPS) in muscle, which
13
involves up-regulation of Atrogin-1/MAFbx and muscle RING-finger protein-1 (MuRF-
14
1) [6], and is considered to be the main mechanism of muscle wasting in humans [7].
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The thiazolidinediones (glitazones, TZDs) are selective ligands of the nuclear
17
transcription factor peroxisome-proliferator-activated receptor γ (PPARγ) that exert
18
specific metabolic action towards increasing insulin sensitivity [8]. They are widely
19
used in the treatment of diabetes mellitus (DM) type 2, but may cause weight gain,
20
which is regarded as unwanted process in this population [9]. However, this might be
21
a favourable effect in patients with cachexia, especially since insulin sensitizers were
22
shown to attenuate lean mass loss in older patients with DM [10]. In a mouse model
23
of cancer cachexia, rosiglitazone (RSG) was shown to reduce weight loss [11, 12].
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It has to be considered, however, that the effects of TZDs on heart function are
26
somewhat controversial. Therapy with TZDs results in increased fluid retention [13], 5
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which has led the authorities to discourage the use of TZDs in heart failure patients
2
[14]. Alternatively, echocardiographic studies showed that TZDs after 52 weeks of
3
therapy do not negatively affect myocardial structure or function [15]. RSG was
4
shown to induce eccentric cardiac hypertrophy, associated with an increase in
5
myofibrillar protein content and turnover and slightly increased stroke volume in
6
healthy rats [16], which could be favorable in cancer cachexia, where cardiac
7
remodeling and atrophy impair cardiac function [17].
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1
8
We aimed to investigate the effects of rosiglitazone (RSG) on body wasting, body
10
composition and survival in a rat Yoshida AH-130 hepatoma cancer cachexia model.
11
Due to safety concerns, we also aimed to assess the effect of RSG on cardiac
12
function.
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2. Materials and methods
3
2.1. Tumour Model
4
Male Wister Han rats (mean weight 201.6 ± 1.6 g, age 7-8 weeks) were injected
5
intra-peritoneally with 108 Yoshida AH-130 tumour cells as described previously [18].
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2.2. Study design
8
After tumor inoculation rats were randomized into the placebo (n = 49) or RSG 4
9
mg/kg/day (n = 15) group. The control group (n = 10) was injected with saline. Before
10
tumour inoculation, baseline weight, body composition and quality of life indicators
11
were measured (Figure 1). Treatment with placebo (water) or active compound,
12
given per gavage, started one day after tumour inoculation. All staff handling the rats
13
was blinded to treatment allocation. Animals were housed under specific pathogen
14
free
15
cardiac function was analysed and quality of life was assessed once more. Animals
16
were euthanized 14 days after tumour inoculation. Several animals had to be
17
euthanized prematurely for ethical reasons according to prospectively defined criteria
18
of disease burden (Figure 1) [19]. Body weight and body composition were assessed
19
every second day, but the final assessment of body weight and body composition
20
was performed after removal of the tumour. At the end of the study, organs were
21
removed and weighed. All procedures were approved by the local animal ethics
22
committee.
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conditions in groups of three and were monitored twice daily. On day 11,
23 24
2.3. Body composition
7
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Body composition (fat and lean body mass) was analysed with Nuclear Magnetic
2
Resonance spectroscopy (EchoMRI-700, Echo Medical Systems, Houston, TX), as
3
previously described [18].
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2.4. Echocardiography
6
Echocardiography was performed as described before [20]. Briefly, rats were
7
anesthetized using 1.5% isoflurane and laid in supine position on a platform. Body
8
temperature was monitored and maintained at 36–38°C using a heating pad. All hair
9
was removed from the chest using a chemical hair remover. B- and M-mode tracings
10
were recorded and assessed for left ventricular dimensions and function using the
11
high resolution Vevo 770 system (Visual Sonics, Toronto, Canada).
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2.5. Quality of life indicators
14
Quality of life in animals can be assessed by measurement of spontaneous activity
15
and food intake [21]. Rats were housed individually over a period of 24 hours. Food
16
intake was recorded and spontaneous movement was assessed by an infrared
17
monitoring system (Supermex, Muromachi, Tokyo, Japan).
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2.6. Proteasome activity
20
At the end of the study, the proteolytic activity of the proteasome was analysed as
21
described before [19]. Briefly, protein isolated from gastrocnemius muscle were
22
incubated with one of the fluorogenic substrates (Biomol, Hamburg, Germany) to
23
determine
24
methylcoumarin [Z-LLE-AMC]), chymotrypsin-like activity (succinyl-Leu-Leu-Val-Tyr-
25
7-amido-4-methylcoumarin [LLVY-AMC]) or peptidyl-glutamyl-protein-hydrolysing
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trypsin-like
activity
(benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-
8
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activity (benzoyl- Val-Gly-Arg-7-amidocoumarin [Bz-VGR-AMC]), at 37°C for 1 hour
2
followed by 10 minutes on ice. The fluorescence intensity was measured with a
3
fluorometer (Twinkle LB 970, Berthold, Bad Wildbad, Germany) at 360nm excitation
4
and 460nm emission. The activity, expressed as nmol/mg protein/min, was
5
calculated by using free amidomethylcoumarin (AMC) as working standard.
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2.7. Statistics
8
Data were analyzed using GraphPad PRISM 5.0 (GraphPad Software, Inc, La Jolla,
9
CA, USA). Results are shown as mean ± standard error of the mean (SEM). All data
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were tested for normal distribution using the D'Agostino & Pearson omnibus
11
normality test. Between-group comparison was performed using ANOVA followed by
12
Tukey’s tests for data with normal distribution; data with skewed distribution were
13
analysed by Kruskal-Wallace and Dunns test. All statistical tests were two sided and
14
a p-value < 0.05 was considered significant. Survival was tested by Cox proportional
15
hazard analysis. Hazard ratio (HR) and 95% confidence interval (CI) are shown. A p
16
value of < 0.05 was considered significant.
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1 2
3. Results
3
3.1. Survival
5
Treatment with 4 mg/kg/d RSG had no effect on tumour growth (6.3 ± 0.7 and 5.8 ±
6
0.5 x 109 cells for RSG and placebo, respectively). The survival analysis shows a
7
significant benefit from treatment with RSG in AH-130 tumor bearing rats, with a risk
8
reduction of 62% (HR = 0.38, 95% CI: 0.15 – 0.86, p < 0.05; Figure 2). Survival rate
9
at day 14 was 80% in RSG group compared to 48% in placebo group.
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3.2. Body mass and body composition
12
Tumor bearing animals lost weight, but this loss was reduced with RSG treatment
13
(Figure 3). The average loss of body mass per day was 3.93 ± 0.2 g in placebo
14
animals compared to 2.33 ± 0.81 g in RSG treated animals (p < 0.05). Reduction of
15
body mass resulted from both fat and lean mass wasting. The average loss of lean
16
mass per day was 2.93 ± 0.15 g in the placebo group and 2.00 ± 0.55 g in the treated
17
group (p < 0.001). The average loss of fat mass per day was 0.95 ± 0.04 g in the
18
placebo group and 0.49 ± 0.18 g in the treated group (p < 0.001).
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3.3. Organ weight
21
Mass of all organs was significantly lower in cachectic cancer rats (placebo group)
22
when compared to controls (p < 0.001, Table 1). Liver mass, heart mass and mass of
23
skeletal muscles were not significantly different in RSG group when compared to
24
placebo. The mass of white adipose tissue (WAT), brown adipose tissue (BAT) and
25
spleen was significantly higher in RSG group (p < 0.05 versus placebo).
10
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1
3.4. Cardiac function
3
Cardiac function was similar in both groups before tumour inoculation (data not
4
shown), but was improved by treatment with RSG compared to placebo on day 11
5
(Table 2). There was no difference in left ventricular mass, in the diameters of the left
6
ventricle and volumes as well as thickness of posterior wall, both in diastole and
7
systole. However, there was a greater thickness of the septal wall in systole and an
8
improved fractional shortening (41.2% in RSG group vs. 30.5% in placebo group, p <
9
0.01), ejection fraction (62.4% in RSG group vs. 52.5% in placebo group, p < 0.05)
10
and stroke volume (147.3 µL in RSG group vs. 106.8 µL in placebo group, p < 0.01).
11
The result of the improved pumping function, i.e. systolic function, was a significantly
12
higher cardiac output. The diastolic function assessed as the ratio of E-and A-wave
13
of the mitral blood flow was within the normal range in both groups and not
14
statistically different.
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3.5. Quality of life
17
Food intake and spontaneous locomotor activity were not different before inoculation
18
of the tumor cells. On day 11 there was a significant improvement of food intake by
19
RSG (126%) compared to placebo (7.93 ± 2.09 vs. 4.30 ± 0.48 g/24h, respectively, p
20
= 0.01). Locomotor activity was also higher in RSG (41%) treated animals 41,543 ±
21
5,327 vs. 29,509 ± 1,775 beam breaks/24h, p < 0.05).
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3.6. Proteasome activity
24
Peptidyl-glutamyl-protein-hydrolysing, trypsin-like and chymotrypsin-like activities
25
were all increased in the gastrocnemius muscle of tumor-bearing animals. RSG
11
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significantly reduced the peptidyl-glutamyl-protein-hydrolysing activity (319 ± 28
2
nmol/mg protein/min vs. 167 ± 62 nmol/mg protein/min, p < 0.05) and trypsin-like
3
activity (179 ± 33 nmol/mg protein/min vs. 88 ± 40 nmol/mg protein/min, p < 0.05) in
4
cachectic rats compared to placebo. RSG did not affect the chymotrypsin-like activity
5
(Figure 4).
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4. Discussion
8
We have shown for the first time that RSG improves survival and quality of life in rats
9
with cancer cachexia along with having favorable effects on cardiac function.
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10
Improved survival of rats is probably the result of weight loss prevention, since weight
11
loss is long known to be associated with impaired survival in cancer patients [1].
12
RSG was shown before to reduce the weight loss in tumor-bearing mice. However, it
14
was effective in preventing the weight loss in early cachexia, but had no effect in
15
slowing the rate of weight loss in late-stage cachexia. [11, 12]. Time dependent
16
manner of body weight and adipose tissue loss in rats with cancer cachexia was also
17
shown by Batista et al., who observed significant loss no earlier than day 14 after
18
tumor inoculation [22]. In our study, weight was measured only at baseline and at the
19
end of the study, so we could not observe the time course of weight loss. However,
20
the mechanisms of weight loss may have changed trough the course of the disease.
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22
With regard to body composition, RSG decelerated the loss of both fat and lean
23
mass. In cancer cachexia, fat tissue precedes lean tissue loss and is related to
24
reduction of adipocyte size, not number. Lipolysis is increased and so is the turnover
25
of glycerol and free-fatty acids; however, the exact mechanism of fat tissue loss
12
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remains unknown [23, 24]. PPARγ expression, which is essential for adipocyte
2
differentiation, proliferation, fatty acid uptake and storage, is reduced in WAT [22].
3
TZDs induce WAT lipolysis but also increase the uptake of free-fatty acids by WAT
4
and improve the response of WAT lipolysis to the inhibitory action of insulin [25]. In
5
DM type 2 patients, RGS was shown to contribute to redistribution of regional body
6
fat [26, 27]. In our study, RSG prevented the loss of fat mass in cancer cachexia,
7
which was confirmed with both body composition determination and measurement of
8
WAT and BAT mass at the end of the study. Same was observed in the recently
9
published studies of Asp et al. [11, 12].
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Body composition measurement also revealed the beneficial effect of RSG on lean
12
mass wasting; however, the organ weights of skeletal muscle, heart and liver – while
13
higher - were not significantly different in RSG group compared to placebo. A similar
14
lack of effect was observed in studies of Asp et al. [11, 12]. In skeletal muscle,
15
cancer cachexia increases the expression of Atrogin-1 and MuRF-1, which leads to
16
increased protein degradation. Concomitantly, myofibrillar protein synthesis is
17
reduced [28]. Although we did not show beneficial effect of RSG on skeletal muscle
18
mass, RSG reduced the activity of proteasome system in gastrocnemius muscle.
19
RSG evidently decelerates muscle protein degradation, which could be the
20
consequence of IR improvement. In the study of Wang et al., IR was shown to
21
accelerate muscle protein degradation and RSG was able to reduce it by lowering
22
the expression of Atrogin-1/MAFbx and MuRF1. However, only partial recovery of the
23
cross-sectional area and muscle mass was achieved. It was suggested that the
24
recovery was not complete because RSG did not stimulate protein synthesis [7]. The
25
effect of RSG may also change trough the progression of cachexia. Asp et al.
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1
showed that RSG reduced gene expression of Atrogin-1 and MuRF-1 in early
2
cachexia but not in late-stage cachexia [12].
3
Besides tissue wasting in cancer cachexia, heart function also deteriorates. In our
5
study, rats with cancer had reduced left ventricular ejection fraction, fractional
6
shortening, stroke volume, cardiac output, and other abnormalities. Reduced cardiac
7
contractile function in cancer cachexia results from cardiac remodeling and atrophy.
8
Fibrosis, disrupted alignments of sarcomeric structure and impaired integrity of
9
mitochondria can be seen in cardiac muscle cells [29]. Cardiomyocyte function is
10
depressed during both cellular contraction and relaxation [30].On molecular level,
11
myocardium protein degradation is increased with rise in expression of MuRF-1 and
12
Atrogin-1 and increased level of protein ubiquitination. Additionally, the expression of
13
glucose transporter type 4 (GLUT4) is decreased [29].
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In our study, RSG improved cardiac function in rats with cancer cachexia. It
16
increased left ventricular ejection fraction (LVEF) and cardiac output in comparison to
17
rats receiving placebo. Festuccia et al. have shown that in healthy rats, RSG induces
18
eccentric cardiac hypertrophy, reduces heart rate and slightly increases stroke
19
volume. It switches from fatty acids to glucose as a major source of energy substrate
20
and causes increased turnover of myofibrillar proteins: it increases synthesis,
21
enhances calpain-mediated myofibrillar degradation, but reduces protein degradation
22
through the UPS [25]. In our study, however, a hypertrophy of the heart muscle was
23
not detected - the heart mass and mass of left ventricle were not significantly higher
24
in the RSG group when compared to placebo.
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In vitro studies showed that activation of PPARγ receptors inhibits malignant cell
2
proliferation and suppresses angiogenic tumor phenotype [31, 32]. In rats, TZD were
3
shown to suppress carcinogenesis [33, 34], although the results are somewhat
4
contradictive [35]. Additionally, recent meta-analysis of clinical data showed reduced
5
risk of liver cancer in DM type 2 patients receiving TZD [36]. In our study, however,
6
RSG did not affect the tumor growth, since the number of the tumor cells did not
7
differ significantly between placebo and RSG group.
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8
In the light of translating the findings of our study to human population, one must
10
consider the increased risk of cardiovascular events in patients, treated with RSG
11
that has already led to withdrawal of the drug from the market. In order make the
12
drug applicable to cancer patients, the benefits of attenuated weight loss should
13
outweigh the increased risk for cardiovascular events. Further studies are needed to
14
address this issue.
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5. Limitations
17
The body weight and body composition of the rats were recorded every two days;
18
however, due to the tumor growth, we were not able to determine if weight loss and
19
changes in body composition occurred in a time dependent manner. Measurement
20
of insulin sensitivity could have provided more detailed insight in the molecular
21
mechanisms of RSG effects.
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6. Conclusion
24
In conclusion, RSG improves survival, quality of life and decelerates weight loss in
25
the rat model of cancer cachexia, which could be useful in cancer patients. Despite
15
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discouragement of TZD use in heart failure, RSG could have beneficial effects in
2
cancer patients, where heart function is worsened by cancer cachexia.
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1 2 3
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medical need: facts and numbers. J Cachexia Sarcopenia Muscle 2010;1(1):1-
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3. Wallengren O, Lundholm K, Bosaeus I. Diagnostic criteria of cancer cachexia:
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9. Dormandy JA, Charbonnel B, Eckland DJ, Erdmann E, Massi-Benedetti M, Moules IK, et al.; PROactive investigators. Secondary prevention of
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13. Hernandez AV, Usmani A, Rajamanickam A, Moheet A. Thiazolidinediones
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14. McMurray JJ, Adamopoulos S, Anker SD, Auricchio A, Böhm M, Dickstein K, et al.; ESC guidelines for the diagnosis and treatment of acute and chronic
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16. Festuccia WT, Laplante M, Brûlé S, Houde VP, Achouba A, Lachance D, et al.
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Evidence for a novel serum factor distinct from zinc alpha-2 glycoprotein that
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24. Rydén M, Arner P. Fat loss in cachexia--is there a role for adipocyte lipolysis? Clin Nutr 2007;26(1):1-6.
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25. Festuccia WT, Laplante M, Berthiaume M, Gélinas Y, Deshaies Y. PPARgamma agonism increases rat adipose tissue lipolysis, expression of
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Diabetologia 2006;49(10):2427-36.
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27. Nam JS, Nam JY, Yoo JS, Cho M, Park JS, Ahn CW, et al. The effect of
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rosiglitazone on insulin sensitivity and mid-thigh low-density muscle in patients
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with Type 2 diabetes. Diabet Med 2010;27(1):30-6. 21
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28. White JP, Baynes JW, Welle SL, Kostek MC, Matesic LE, Sato S, et al. The regulation of skeletal muscle protein turnover during the progression of cancer
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cachexia in the Apc(Min/+) mouse. PLoS One 2011;6(9):e24650.
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29. Tian M, Asp ML, Nishijima Y, Belury MA. Evidence for cardiac atrophic
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30. Xu H, Crawford D, Hutchinson KR, Youtz DJ, Lucchesi PA, Velten M, et al.
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Myocardial dysfunction in an animal model of cancer cachexia. Life Sci
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31. Hatton JL, Yee LD. Clinical use of PPARgamma ligands in cancer. PPAR Res
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32. Panigrahy D, Shen LQ, Kieran MW, Kaipainen A. Therapeutic potential of
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33. Borbath I, Leclercq I, Moulin P, Sempoux C, Horsmans Y. The PPARgamma
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34. Shen B, Chu ES, Zhao G, Man K, Wu CW, et al. PPARgamma inhibits
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35. Lubet RA, Fischer SM, Steele VE, Juliana MM, Desmond R, Grubbs CJ. Rosiglitazone, a PPAR gamma agonist: potent promoter of
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36. Wang F, Zhao SZ, Zhang MY, Ma YL, Zhang P, Qin HL. Decreased risk of liver cancer with thiazolidinediones therapy in patients with type 2 diabetes:
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results from a meta-analysis. Hepatology 2013 Jan 16. doi:
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10.1002/hep.26259. [Epub ahead of print]
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37.
2
FIGURE LEGENDS
3
Figure 1. Flow chart of experimental protocol.
4
D = day, QoL = quality of life, Echo = echocardiography
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Figure 2. Survival analysis. RSD improves survival in tumor-bearing rats.
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Figure 3. Changes in body weight and body composition. There were no differences
9
between the groups at baseline. The profound loss of body weight of bearing rats
10
was attenuated by RSG. A similar effect was seen for lean mass and fat mass. * p <
11
0.05, ** p < 0.01, *** p < 0.001: baseline versus day 14; # p < 0.05, ## p < 0.01, ###
12
p < 0.001: control/RSG versus placebo on day 14
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Figure 4. Proteasome activity. RSG reduces peptidyl-glutamyl-protein-hydrolysing
15
(PGPH) and trypsin-like activity, but not chymotrypsin-like activity of the proteasome.
16
* p < 0.05: RSG/control versus placebo
17
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Table 1: Organ weight Placebo
Rosiglitazone
(n = 10)
(n = 49)
4mg/kg/d (n = 15)
liver [mg]
10090±300***
6228±186
6723±481
spleen [mg]
633±26***
187±12
WAT [mg]
1348±90***
93±26
332±173*
BAT [mg]
279±23***
89±4
123±22*
GC [mg]
1179±30***
725±17
tibialis [mg]
428±7***
265±6
soleus [mg]
94±3***
EDL [mg]
100±2***
heart [mg]
751±8***
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Control
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252±40*
794±55 289±17
70±1
66±4
64±1
67±4
506±12
526±26
WAT = white adipose tissue, BAT = brown adipose tissue, GC = gastrocnemius, EDL = extensor
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* p < 0.05, *** p < 0.001 versus placebo
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Table 2: Cardiac function Placebo
Rosiglitazone
(n = 10)
(n = 40)
4 mg/kg/d (n = 15)
LVEF [%]
67.8 ± 2.5*
52.5 ± 2.0
62.4 ± 2.0*
LVFS [%]
50.4 ± 2.1**
30.5 ± 1.7
LVEDD [mm]
6.37 ± 0.11*
5.74 ± 0.12
LVESD [mm]
3.44 ± 0.19*
3.98 ± 0.11
LVEDV [µL]
269.4 ± 14.5**
199.9 ± 9.8
236.0 ± 16.7
LVESV [µL]
85.5 ± 6.0
93.1 ± 4.9
88.7 ± 7.5
LVSV [µL]
183.9 ± 13.4***
Heart rate [bpm]
413 ± 10
CO [mL/min]
78.85 ± 6.57***
LVPW dia [mm]
1.86 ± 0.13
LVPW sys [mm]
2.96 ± 0.15***
Septum dia [mm]
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Control
41.2 ± 2.1** 6.16 ± 0.17
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3.61 ± 0.15
147.3 ± 11.8**
375 ± 13
371 ± 9
41.97 ± 2.97
56.38 ± 5.74*
1.68 ± 0.05
1.53 ± 0.06
2.18 ± 0.06
2.22 ± 0.07
1.56 ± 0.07
1.47 ± 0.04
1.32 ± 0.04*
Septum sys [mm]
2.82 ± 0.09***
2.15 ± 0.05
2.40 ± 0.07*
LV mass [mg]
574 ± 42***
430.5 ± 11.3
420.2 ± 19.2
1.57 ± 0.19
1.36 ± 0.09
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E/A
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106.8 ± 6.8
1.57 ± 0.10
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LV = left ventricular, EF = ejection fraction, FS = fractional shortening, EDD = end-diastolic diameter, ESD = end-systolic diameter, EDV = end-diastolic volume, ESV = end-systolic volume, SV = stroke volume, CO = cardiac output, PW = posterior wall, dia = diastole, sys = systole * p < 0.05, ** p < 0.01, *** p < 0.001 versus placebo
ACCEPTED MANUSCRIPT
Sacrificed animals
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n=3
n = 15
D11
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Tumor inoculation
D0
Placebo group
n = 26
EP
n = 49
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Body weight Body composition Echo QoL
n = 12
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D1
STUDY END D14
D1
n = 23 Sacrificed animals
D11
D14
Echo QoL
Body weight Body composition Organ weight
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SC
Survival proportions [%]
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100 80 60
20 0 4
6
8
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2
10
12
days after tumor inoculation
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40
14
4m g/kg/d rosiglitazone (n=15) placebo (n=49)
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body weight [g]
80 60
300 ###
40
***
***
###
**
20 #
0 -20
100
-40 -60
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200
delta body weight [g]
control
0 14 control
0 14 0 14 placebo rosiglitazone
SC
0
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lean mass [g] 250
rosiglitazone
delta lean mass [g]
60
**
***
***
###
40
###
200 150
placebo
###
20
#
0
TE D
100 50 0
AC C
30 ###
***
***
#
-60
0 14 0 14 placebo rosiglitazone
fat mass [g]
20
-40 control
placebo
rosiglitazone
EP
0 14 control
-20
delta fat mass [g] 15 ###
10 5
* #
10
0 -5 -10
0
0 14 control
0 14 0 14 placebo rosiglitazone
###
-15 control
placebo
rosiglitazone
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PGPH activity
300
* 200
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nmol/mg protein/min
400
100 0 placebo
rosiglitazone
SC
control
200 150
*
*
100
TE D
nmol/mg protein/min
250
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trypsin-like activity
50 0
placebo
rosiglitazone
EP
control
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chymotrypsin-like activity
nmol/mg protein/min
30
20
10
0 control
placebo
rosiglitazone