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Year: 2010
Erythropoietin treatment leads to reduced blood glucose levels
and body mass: Insights from murine models
Katz, O; Stuible, M; Golishevski, N; Lifshitz, L; Tremblay, M; Gassmann, M;
Mittelman, M; Neumann, D
Katz, O; Stuible, M; Golishevski, N; Lifshitz, L; Tremblay, M; Gassmann, M; Mittelman, M; Neumann, D (2010).
Erythropoietin treatment leads to reduced blood glucose levels and body mass: Insights from murine models. The
Journal of Endocrinology:Epub ahead of print.
Postprint available at:
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Posted at the Zurich Open Repository and Archive, University of Zurich.
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Originally published at:
The Journal of Endocrinology 2010, :Epub ahead of print.
Erythropoietin treatment leads to reduced blood glucose levels
and body mass: Insights from murine models
Abstract
Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into
erythrocytes. The last decade has revealed non-renal sites of EPO production and extrahematopoietic
expression of the EPO-receptor (EPO-R), thus suggesting that EPO has pleiotropic functions. Here we
addressed the interplay between EPO / glucose metabolism / body weight, by employing a panel of
relevant experimental murine models. The models focused on situations of increased EPO levels,
including EPO injected C57BL/6 and BALB/c mice, as well as transgenic mice (tg6) constitutively
overexpressing human EPO, thus exposed to constantly high EPO serum levels. As experimental models
for diabetes and obesity, we employed protein Tyr phosphatase 1B (PTP1B) knockout mice associated
with resistance to diabetes (PTP1B-/-), and ob/ob mice susceptible to diabetes and obesity. The data
presented herein demonstrate EPO-mediated decrease in blood glucose levels in all mice models tested.
Moreover, in the ob/ob mice we observed EPO-mediated attenuation of body weight gain and reduction
of hemoglobin A1C. Taken together our data bear significant clinical implications of EPO treatment in
the management of a wide range of metabolic diseases, thus adding an important novel therapeutic
potential to this pleiotropic hormone.
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Manuscript submitted for review to Journal of Endocrinology
Katz et al., Erythropoietin mediated reduction in blood glucose
Erythropoietin treatment leads to reduced blood glucose levels and
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body mass: Insights from murine models
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Odelia Katz , Matthew Stuible , Nathalia Golishevski , Lilach Lifshitz , Michel L.
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Tremblay , Max Gassmann , Moshe Mittelman and Drorit Neumann
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Department of Cell and Developmental Biology, Department of Medicine A,
Tel Aviv Sourasky Medical Center, Tel Aviv 64239; 1,2Sackler Faculty of
Medicine, Tel Aviv University, Tel Aviv 69978, Israel; 3Goodman Cancer Centre
and Department of Biochemistry, McGill University, 1160 des Pins West,
Montreal, QC, Canada, H3A 1A3; 4Institute for Veterinary Physiology, Vetsuisse
Faculty and Zurich Center for Integrative Human Physiology (ZIHP), University
of Zurich, 8057 Zurich, Switzerland
Key words: erythropoietin, murine models, glucose, metabolism
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Abbreviations: EPO, erythropoietin; EPO-R, erythropoietin receptor; rHuEPO,
recombinant human erythropoietin
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Running Title: Erythropoietin mediated reduction in blood glucose
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Moshe Mittelman M.D.
Address: Department of Medicine, Sourasky Medical Center, 6 Weizmann St.
64239 Tel Aviv, Israel
Phone: +972-3-6973366
Fax: +972-3-6974855
E-mail: [email protected]
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*Corresponding authors:
Drorit Neumann Ph.D.
Address: Cell and Developmental Biology, Sackler Faculty of Medicine
Tel Aviv University, 69978 Tel Aviv, Israel
Phone: +972-3-6407256
Fax: +972-3-6407432
E-mail: [email protected]
[email protected]
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Manuscript submitted for review to Journal of Endocrinology
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Katz et al., Erythropoietin mediated reduction in blood glucose
Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor
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cells into erythrocytes. The last decade has revealed non-renal sites of EPO
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production and extrahematopoietic expression of the EPO-receptor (EPO-R), thus
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suggesting that EPO has pleiotropic functions. Here we addressed the interplay
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between EPO / glucose metabolism / body weight, by employing a panel of relevant
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experimental murine models. The models focused on situations of increased EPO
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levels, including EPO injected C57BL/6 and BALB/c mice, as well as transgenic mice
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(tg6) constitutively overexpressing human EPO, thus exposed to constantly high EPO
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serum levels. As experimental models for diabetes and obesity, we employed protein
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Tyr phosphatase 1B (PTP1B) knockout mice associated with resistance to diabetes
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Abstract
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-/-
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herein demonstrate EPO-mediated decrease in blood glucose levels in all mice models
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tested. Moreover, in the ob/ob mice we observed EPO-mediated attenuation of body
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weight gain and reduction of hemoglobin A1C. Taken together our data bear
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significant clinical implications of EPO treatment in the management of a wide range
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of metabolic diseases, thus adding an important novel therapeutic potential to this
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pleiotropic hormone.
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Introduction
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(PTP1B ), and ob/ob mice susceptible to diabetes and obesity. The data presented
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The hematopoietic growth factor Erythropoietin (EPO) is produced in the kidney in
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response to hypoxia, and stimulates erythropoiesis in the bone marrow (Krantz 1991;
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Spivak, et al. 1991). Recombinant human EPO (rHuEPO) is an effective treatment for
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anemia of various etiologies, including anemias associated with renal failure
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(Eschbach, et al. 1989 ; Winearls, et al. 1986) and cancer-related (Mittelman 1996;
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Spivak 1994). The presence of EPO receptors (EPO-R) on cells other than erythroid
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progenitors, including endothelial, myocardial, neural and retinal cells, suggests that
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EPO has other biological functions in addition to erythropoiesis, reviewed in (Arcasoy
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2008). Hence, such functions include neuroprotection (Brines, et al. 2004; Cilloni, et
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al. 2004), anti-neoplastic activity (Katz, et al. 2005; Mittelman, et al. 2001),
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improvement in congestive heart failure (Camici, et al. 2007; Fiordaliso, et al. 2005;
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Silverberg, et al. 2002). We (Ghezzi and Mengozzi 2007; Lifshitz, et al. 2009;
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Prutchi-Sagiv, et al. 2006a; Prutchi-Sagiv, et al. 2006b; Prutchi-Sagiv, et al. 2008), as
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well as others (Ghezzi and Mengozzi 2007), have found EPO associated improvement
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in immunological functions.
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In that respect, we have been attracted by the reports on EPO effects on glucose
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metabolism in haemodialysis patients (Allegra, et al. 1996; Borissova, et al. 1993),
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that are associated with elevated insulin sensitivity (Spaia, et al. 2000; Tuzcu, et al.
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2004). EPO effects were also documented on the metabolism of proteins and lipids;
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however, the latter are still controversial, reviewed in (Allegra, et al. 1997).
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clinical practice and is especially relevant in subjects with metabolic syndrome.
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Metabolic syndrome is common, affecting more than 30% of adults in the US
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(Feldeisen and Tucker 2007), and is referred to as a “cluster of metabolic
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abnormalities”, including obesity, hyperglycemia, dyslipidemia and hypertension
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(Chew, et al. 2006). In the 'modern' world, diet and lifestyle contribute to the high
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incidence of obesity which is linked to increased risk for diabetes, cancer and
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cardiovascular diseases, that leads to a reduction in life expectancy (Kolonin, et al.
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2004). In the last few decades the prevalence of obesity and diabetes has been
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increasing rapidly, reviewed in (Keller 2006). Consequently, the concern regarding
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obesity and its metabolic implications has directed intensive scientific effort
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The ability to control glucose, protein and lipid metabolism is of paramount value in
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concerning the adipose tissue, which is a major endocrine and signaling organ
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involved in a wide range of physiological responses, reviewed in (Rosen and
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Spiegelman 2006).
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The positive effect of rHuEPO treatment on glucose metabolism in hemodialyzed
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patients set the scene for exploring critically the interplay between EPO / glucose
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metabolism / body weight, in the absence of any clinical problems. We have thus
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employed a panel of relevant murine models to investigate this issue. The models
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focused on situations of increased EPO levels, including EPO injected C57BL/6 and
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BALB/c mice, as well as transgenic mice constitutively overexpressing human EPO
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in an oxygen independent manner (tg6; (Ruschitzka, et al. 2000; Vogel, et al. 2003)).
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EPO plasma levels in these mice are typically 10-12 fold higher than wt EPO levels
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(under normoxic conditions) (Ruschitzka et al. 2000; Vogel et al. 2003). This
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elevation causes a doubling of their hematocrit from 40 to 80-90 % and up to 25% of
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the body weight is directed to the blood (Ruschitzka et al. 2000; Vogel et al. 2003).
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Expression of human EPO in these mice has been detected in the brain (where it most
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probably acts locally) as well as in the lung, from where it reaches circulation
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(Heinicke, et al. 2006). As experimental models for diabetes and obesity, we
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employed PTP1B knockout mice (Elchebly, et al. 1999) associated with resistance to
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diabetes (PTP1B-/-), and ob/ob mice susceptible to diabetes and obesity, reviewed
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(Houseknecht and Portocarrero 1998). Taken together, the data presented herein,
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weight gain, thus bearing significant clinical implications in the management of a
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wide range of metabolic diseases.
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demonstrate EPO-associated decrease in blood glucose levels and attenuation of body
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Materials and methods
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Mice
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Female mice of the inbred strains BALB/c and C57BL/6, aged 8 and 12 weeks,
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respectively, were obtained from the Tel-Aviv University Animal Breeding Center.
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Transgenic mice overexpressing HuEPO (tg6; (Ruschitzka et al. 2000)) aged 3-6
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months were bred by us, and their wt littermates (C57BL/6) were used as controls.
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Male ob/ob mice [C57BL/6], aged 7 weeks, were obtained from Harlan (USA). Three
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month old male PTP1B-/- mice [BALB/c] were previously described (Elchebly et al.
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1999). Mice were maintained on a standard rodent chow diet (Koffolk, Israel). All
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procedures were approved by the animal care committees at the Sackler Faculty of
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Medicine Tel-Aviv University, Israel and at McGill University, Canada.
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Injected materials
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weekly injections (s.c.) of 180 U rHuEPO or diluent (control), for the duration of the
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experiment. Glucose – D glucose (Sigma, USA). Insulin – Actrapid HM(ge) (Novo
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Nordisk, Israel).
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Food consumption
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EPO - EPREX , rHuEPO, Epoetin alfa (Cilag Ltd, Switzerland). Mice received three
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Food consumption was measured during four weeks. Pre-weighed food was provided
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to the mice, the remaining food was measured subsequently, and the average food
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consumption per week was calculated for each mouse.
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Metabolic assays
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Serum glucose levels were determined by Accu-check go glucometer (Roche,
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Germany). Hemoglobin A1C (HbA1c) concentration in the blood was measured with
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DCA2000 analyzer (Bayer, USA). Serum insulin levels were measured using a
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commercial enzyme immunoassay kit (Mercodia, Sweden).
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Glucose tolerance test (GTT) – mice deprived of food for over four hours received an
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intraperitoneal injection of glucose (2 g/kg body weight). Blood samples were then
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obtained from the tail for glucose determination at the indicated times.
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Results are expressed as the mean ± standard error of the mean (SEM). All results
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were analyzed by Student's unpaired two-tailed t-test. Values of P < 0.05 were
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considered statistically significant.
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Statistical analysis
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Results
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10-12 fold elevated EPO serum levels on blood glucose level EPO overexpressing
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transgenic mice (tg6) (Ruschitzka et al. 2000). As shown in Fig. 1A, tg6 mice are
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hypoglycemic as compared to their normal (wt) littermates. Hence, in the fed state,
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tg6 mice displayed 49% less glucose level in their blood compared to the wt. This
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finding raised the question of whether short exposure to EPO may also affect the
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blood glucose levels. We thus compared the effect of rHuEPO treatment on blood
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glucose levels in murine models that are associated with diabetes and obesity (Fig. 1B
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and C), as well as wt mice (Fig. 1D and E). In these experiments, mice were injected
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with rHuEPO (180U), three times weekly on alternating days. We found that after one
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We first examined, under non-fasting conditions, the effect of constitutively
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levels were further reduced during the second week of treatment. Moreover, this
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reduction was also observed within the group of rHuEPO-treated mice as compared to
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their initial blood glucose levels.
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week of treatment, all rHuEPO-treated mice had lower blood glucose levels, and these
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tolerance test (GTT) in all mice (Fig. 2). In rHuEPO-treated mice the test was
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performed during the third week of treatment. Administration of a bolus of glucose to
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tg6 and rHuEPO-treated mice resulted in a more rapid clearance of glucose than was
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observed in wt and the control group receiving just the diluent, instead of rHuEPO. In
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tg6 mice, a prominent hypoglycemia was evident 15 min following the glucose
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injection.
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To investigate the effect of EPO on glucose clearance, we performed a glucose
In order to determine whether tg6 mice are particularly sensitive to insulin, we
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measured serum insulin levels at fed state, and performed intraperitoneal insulin
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tolerance test (Fig. 3). The results show that tg6 mice have lower serum insulin levels
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compared to their wt littermates (Fig. 3A). It thus appears that tg6 mice are more
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insulin sensitive, because they maintained lower glucose levels with significantly
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reduced insulin concentrations. This was further substantiated as based on the results
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of the insulin tolerance test. The results indicated a hypoglycemic response in tg6
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mice 15 min after insulin injection, a state that was evident at all time points. In
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contrast, wt mice had hyperglycemic response at 15 min, followed by a hypoglycemic
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response at the later time points (Fig. 3B).
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metabolism, using HbA1c measurements. This test was based on the fact that in
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patients with diabetes, HbA1c levels reflect the blood glucose levels during the
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preceding period of several months (Rohlfing, et al. 2002). We thus measured HbA1c
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Based on these data, we assessed the ability of EPO to affect the glucose
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circulating erythrocytes in mice, the test was performed in ob/ob mice during the third
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week of rHuEPO treatment. HbA1c was reduced by 18% and 15% in tg6 and
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rHuEPO-treated ob/ob mice, respectively, compared to their relevant control groups.
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Furthermore, the decrease in ob/ob mice was evident already after a short rHuEPO
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treatment.
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in tg6 and rHuEPO-treated ob/ob mice (Fig. 4). Due to the rapid half life of
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measured the body weight of tg6 mice as compared to their wt littermates. As shown
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in Fig. 5A, the body weight of tg6 mice was lower than that of wt mice, in both males
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and females. To determine whether these weight differences result from a decrease in
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food intake, we estimated the amount of food consumed by the mice (Fig. 5B). The
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results showed that food intake was actually higher in tg6 mice than in wt mice.
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Hence, tg6 mice maintained low body weight, even when higher amounts of food
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were consumed. To further resolve the differences in body weight we analyzed the
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We next questioned whether EPO has any effect on the body mass. We
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weights of distinct tissues, including liver, muscle (gastrocnemius), epididimal fat and
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spleen tissue (Fig. 6). Although histological examination of these organs revealed no
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overt differences, we found that tg6 mice had 42% less epididimal fat mass compared
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to their wt littermates. Differences in adipose tissues were also noted in the
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subcutaneous skin fat; tg6 mice had very thin skin with a thin layer of subcutaneous
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fat. However, the blood lipids profile of the tg6 mice did not differ from those of the
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wt littermates (data not shown).
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we examined whether rHuEPO treatment has any effect on the body mass in ob/ob
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mice. We thus measured the body weight gain and food consumption of ob/ob mice
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during two weeks of rHuEPO treatment (Fig. 7). The results indicated that rHuEPO-
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To evaluate the clinical significance of these EPO effects on the body mass,
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while non-treated ob/ob mice had 24% weight gain, the rHuEPO-treated ob/ob mice
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had only 17% weight gain (p<0.004), in spite of the fact that there was no difference
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in food intake between these groups.
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treated ob/ob mice gained less weight compared to the non-treated ob/ob mice. Hence,
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insulin sensitivity and body mass, pointing to potential benefits to diabetic and obese
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patients.
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Taken together, our data indicate that EPO has effects on glucose homeostasis,
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Discussion
The present study deals with the emerging concept that EPO is a pleiotropic 238
hormone, acting also beyond its role in erythropoiesis (Krantz 1991). Previous reports 239
have documented an effect of EPO on reduction of blood glucose levels (Allegra et al. 240
1996; Cayla, et al. 1999; Rasic-Milutinovic, et al. 2008). Most of these studies were 241
performed on patients suffering from diabetes, a condition linked to insulin resistance 242
(Pothiwala, et al. 2009). Long-term rHuEPO therapy was found to improve glucose 243
metabolism in maintenance hemodialysis patients, mainly by reduction of insulin 244
resistance (Allegra et al. 1996). In rats EPO treatment was also associated with lowered 245
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blood glucose levels, both in resting and under physical stress (Cayla et al. 1999).
In the current study we explored the effect of EPO on glucose metabolism by 247
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employing a panel of murine experimental models. Each of these models enables a 248
unique analysis of the in-vivo effects of EPO on blood glucose and/or body weight. We 249
measured these parameters under constant exposure to high EPO levels, as well as 250
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under short term exposure to EPO (EPO-injected mice). The transgenic murine model 251
tg6, that overexpresses human EPO (Ruschitzka et al. 2000) was thus employed to 252
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determine the effects of long term, constant exposure to high EPO levels. The EPO 253
injected mice included (a) normal – wild type C57BL/6 and BALB/c mice; (b) PTP1B-/- 254
mice that are resistant to both diabetes and obesity (Elchebly et al. 1999; Klaman, et al. 255
2000); (c) ob/ob mice as a model for obesity and diabetes (Muzzin, et al. 1996; 256
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Pelleymounter, et al. 1995).
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The results showed that the tg6 mice that are continuously exposed to EPO were 258
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hypoglycemic, even under non-fasting conditions. This result gained support from the 259
experiments in which EPO was administered to mice for a period of 2 weeks. 260
Moreover, the effect of EPO on the reduction of glucose levels was evident already one 261
week following EPO administration in all the experimental models tested, including the 262
PTP1B-/- and ob/ob mice. This conclusion gained further support by the metabolic tests, 263
namely GTT. EPO overexpression or administration led to improved glucose clearance 264
in all the tested murine models. Finally, HbA1c measurements provide a surrogate 265
marker for blood glucose levels during several months prior to the test and are routinely 266
employed clinically for the management of diabetes (Giugliano, et al. 2008; Rohlfing et 267
al. 2002). In that respect, HbA1c levels were reduced in the rHuEpo treated ob/ob mice, 268
thus pointing to the clinical relevance of EPO treatment to improve glucose metabolism 269
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in diabetic subjects. The fact that the decrease in HbA1c was observed as early as three 270
weeks following EPO administration, could result from the rapid metabolism 271
characteristic of the mice (Wickler and Gleeson 1993), thereby differing from the 272
profile of HbA1c response in human subjects (Bloomgarden 2006). Notably, the 273
decrease in HbA1c in the tg6 mice was in the same range as in the EPO injected ob/ob 274
mice, demonstrating that long term as well as short term exposures to EPO had a 275
similar effect on this parameter, manifested in sustained levels of the HbA1c within the 276
normal range.
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What could be the mechanisms of EPO effects on blood glucose levels?
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Lowered blood glucose associated with exposure to high EPO levels may result from
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Hagen, et al. 2009). At least two adaptive mechanisms allow the tg6 mice to cope
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with this excessive erythrocytosis: highly elevated NO expression as well as a reduced
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erythrocyte life span, the latter keeping the erythrocytes young and flexible and thus,
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preventing the blood from becoming too viscose (Bogdanova, et al. 2007; Ruschitzka
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et al. 2000; Vogel et al. 2003). Nevertheless, tg6 mice live only for about a year and
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die due to multiple organ degeneration (Heinicke et al. 2006).
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Analysis of the ventilatory response in hypoxia revealed that both cerebral and
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circulating EPO enhanced ventilation in hypoxic tg6 male and female mice (Soliz, et
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al. 2007; Soliz, et al. 2009). Interestingly, when measuring body temperature, oxygen
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consumption and carbon dioxide production under normoxia and hypoxia conditions
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neither of these parameters was changed compared to the wt control littermates,
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suggesting that the general metabolic drive was not changed.
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an increase in the erythrocyte counts and their consequent uptake of glucose (Montel-
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Another possibility to be considered, although not necessarily mutually exclusive is
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that EPO may also operate via increasing sensitivity to insulin. This is in line with our
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finding that the tg6 mice were more responsive to insulin and thus maintained lower
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glucose levels under lower levels of insulin, and insulin injection resulted actually in
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hypoglycemia (Fig. 3). EPO-associated increased sensitivity to insulin was not
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evident in the ob/ob mice treated with EPO for 2-3 weeks (data not shown). Hence, it
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is possible that longer exposure to EPO is necessary to mediate this effect in these
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mice. The findings that pancreatic beta-cells harbor functional EPO-Rs and that EPO
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acts directly on them (Fenjves, et al. 2003), raises the possibility that EPO treatment
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may also affect insulin secretion by the pancreatic cells.
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The molecular mechanism by which EPO affects sensitivity to insulin is not yet 303
resolved. EPO may operate via an increase in nitric oxide (NO) (Beleslin-Cokic, et al. 304
2004), which is a powerful vasodilator as well as insulin-sensitizer, reviewed in 305
(Marzo, et al. 2008). Of note tg6 mice express vast amounts of NO (Ruschitzka et al. 306
2000). In addition, the transcription factor CEBP/α was found to decrease upon 307
administration of EPO (Pinto, et al. 2008). This transcription factor was found to be 308
associated with energy homeostasis (Hackett, et al. 1995). Thus for example, CEBP/α-/- 309
mice do not display glycogen stores in the liver, they have elevated EPO-R mRNA in 310
the liver and they die of hypoglycemia shortly following birth (Wang, et al. 1995; 311
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Zhang, et al. 1997). Our observation that liver CEBP/α levels are reduced in the tg6 312
mice (data not shown) is in line with the possibility that the effects of EPO on glucose 313
metabolism may also involve the reduction of this transcription factor.
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Our results in the murine models led us to an insight into possible clinical 315
applications. In that respect, we examined the effect of EPO treatment on the glucose 316
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levels and insulin consumption in a diabetic patient. Indeed, EPO treatment reduced the 317
blood glucose levels and reduced the required doses of insulin (data not shown). These 318
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data are supported by a recent report on the effects of EPO on lowered insulin 319
consumption and HbA1c in a diabetic patient with chronic kidney disease-induced 320
anemia (Brown, et al. 2009). These findings now call for an extensive clinical study, on 321
a larger scale of patients.
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Concomitant with the effects of EPO on glucose levels and insulin sensitivity, we 323
found that EPO administration or overexpression were associated with a decrease in 324
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body weight. This decrease is at least partially associated with reduced amount of fat 325
tissue, especially evident in the leanness of the tg6 mice. Our results are in line with a 326
recent publication demonstrating that targeted over expression of EPO in muscle tissue 327
of mice protected against diet induced obesity and increased metabolic parameters 328
(Hojman, et al. 2009). In line with this observation is our finding that EPO associated 329
attenuated body weight gain in the ob/ob mice, despite the similar food intake as the 330
diluent treated mice. The precise mechanism underlying the decreased body weight has 331
yet to be elucidated. It does not appear to be related to nausea with or without reduced 332
food intake, since this parameter was tested and ruled out. An increased energy 333
consumption or expenditure is thus a possible mechanism, related to the main findings 334
of this study, i.e. reduced blood glucose levels associated with EPO.
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To gain insight into the molecular mediators we questioned whether regulation of 336
PTP1B could explain these EPO mediated effects on glucose metabolism. Our choice 337
to focus on PTP1B leans on the finding that it is a pivotal down-regulator of insulin 338
signaling (Boute, et al. 2003; Elchebly et al. 1999; Issad, et al. 2005; Rondinone, et al. 339
2002; Waring, et al. 2003) and that PTP1B-/- mice are resistant to obesity and diabetes 340
(Elchebly et al. 1999; Haj, et al. 2005; Klaman et al. 2000). Moreover, PTP1B was 341
shown by us (Cohen, et al. 2004), and by others (Callero, et al. 2007), to participate in 342
downregulation of EPO signaling. Our present finding that EPO treatment culminated 343
in reduction of blood glucose in the PTP1B-/- mice, thus rules out the possibility that 344
this EPO action is mediated via PTP1B.
345
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Various aspects of EPO action in rodent models have been documented, including 346
prevention of diabetes-induced podocyte damage (Schiffer, et al. 2008), enhancement 347
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of wound healing (Galeano, et al. 2004) and promotion of vascular cell viability
348
(Chong, et al. 2007). Taken together, our findings single out EPO as a potential novel 349
350
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glucose regulator.
The current study exemplifies an exciting potential application for EPO as an 351
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adjunct anti-diabetic/obesity drug to reduce blood glucose levels and attenuate weight 352
gain especially where these are associated with low levels of hemoglobin. Obviously, 353
introducing EPO for these indications would require additional basic research as well as 354
clinical trials. The idea that EPO acts on glucose metabolism places this hormone in a 355
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new and most important area, with significant clinical applications in diabetes and 356
obesity. These are currently critical health issues at the forefront of medical and social 357
interest.
358
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359
Declaration of interest: There is no conflict of interest that could be perceived as
360
prejudicing the impartiality of the research reported
361
Funding: The project was supported in part by a grant from the Chief Scientist's
362
Office of the Ministry of Health, Israel; by the Israel Cancer Association, by the
363
Israeli Cancer Research Foundation, (DN), and by the Swiss National Science
364
Foundation (MG).
365
[email protected]
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Katz et al., Erythropoietin mediated reduction in blood glucose
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requirements for Ph.D. degree by Odelia Katz, Sackler Faculty of Medicine, Tel Aviv
367
University, Israel.
368
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574
Legends to Figures
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Figure 1: EPO-associated reduction in blood glucose levels
577
(A). Blood glucose was measured in 3 months old tg6 mice and in their wt littermates.
578
These measurements were under non-fasting conditions. (B-E) Murine models were
579
injected with rHuEPO (180 U) or with diluent (control), 3 times weekly on alternating
580
days, for a period of 2 weeks. Glucose measurements were performed before EPO
581
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injections (initial), and one and two weeks following EPO injections. These
582
measurements were performed after 4 h of fasting. White and black squares represent
583
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diluent and EPO injected mice respectively. (B). ob/ob mice, 8 weeks old. (C).
584
PTP1B-/- mice, 3 months old. (D). C57BL/6 mice, 3 months old. (E). BALB/c mice, 8
585
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SEM. *P < 0.02, **P < 0.003, *** P < 0.0001.
587
Figure 2: Improved glucose tolerance conferred by EPO
588
GTTs were performed in the following mouse strains: (A). tg6 and wt littermates
589
iew
weeks old. The number of mice in each group is indicated. Values depict mean ±
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(solid and open squares, respectively). (B). ob/ob. (C). PTP1B-/-. (D). C57BL/6. (E).
590
BALB/c. For the rHuEPO and diluent treated mice, GTTs were performed after 2
591
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weeks of rHuEPO or diluent injections. The number of mice in each group is
592
indicated, solid and open squares indicate EPO and diluent injected mice respectively.
593
Values depict mean ± SEM. * P < 0.03, ** P < 0.003.
594
Figure 3: Lower insulin levels (A) and increased sensitivity to insulin (B) in tg6
595
mice
596
(A). Serum concentrations of insulin in fed tg6 mice and their wt littermates, n = 5
597
mice/group. *P = 0.04 (B) ITT was performed in tg6 mice and their wt littermates.
598
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Katz et al., Erythropoietin mediated reduction in blood glucose
599
insulin administration. n = 4 mice/group. Values depict mean ± SEM. *P = 0.05.
600
Figure 4: EPO-associated reduction in hemoglobin A1c
601
Groups of ob/ob mice were injected with rHuEPO or with diluent (control), three
602
times weekly on alternating days for 3 weeks. Hemoglobin A1c was measured in the
603
sera of the ob/ob mice (n = 5 and 6 for rHuEPO-treated and diluent-treated mice,
604
respectively) and in the tg6 mice and their wt littermates (n = 4 and 3 tg6 and wt mice,
605
respectively) using ELISA. Values depict mean ± SEM. *P < 0.03, **P = 0.001.
606
Figure 5 : Reduced weight despite higher food consumption in tg6 mice
607
Body weight and food consumption of tg6 mice (4 months old), as compared to their
608
wt littermates. (A) Body weight of female tg6 mice and their wt littermates (n = 17
609
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Blood glucose values are expressed as a percentage of glucose concentration, prior to
610
(tg6) and 6 (wt) mice/group), *P = 0.02, **P < 0.004. (B) Weekly food consumption
611
of female and male tg6 mice and their wt littermates, as calculated over a period of 4
612
weeks. Results are expressed as grams of food/mouse/week. n = 5 mice/group. Values
613
depict mean weekly consumption of food per mouse ± SEM. **P < 0.004
614
Figure 6: tg6 mice have less epididymal fat tissue
615
(A). Spleens, livers, muscle (gastrocnemius) and epididymal fat were dissected from
616
female tg6 mice and their wt littermates and were weighed. Organ weights were
617
normalized to total body weight. n = 3 mice/group, values depict mean ± SEM, *P =
618
0.05, **P = 0.014. (B). Photographs of mouse cavities; the arrows indicate the
619
epididymal fat.
620
Figure 7: EPO administration attenuates weight gain in ob/ob mice
621
Weight gain (A) and food consumption (B) of ob/ob mice injected with EPO or with
622
diluent (control). Body weight is presented as percentage change from initial body
623
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(tg6) and 19 (wt) mice/group) and of male tg6 mice and their wt littermates (n = 8
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Katz et al., Erythropoietin mediated reduction in blood glucose
weight. Food consumption of individual ob/ob mice was measured for a period of 2
624
weeks. Results are expressed as grams of food/mouse/week. n = 6 mice/group. Values
625
depict mean ± SEM. * P = 0.03, ** P = 0.003.
626
627
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Manuscript submitted for review to Journal of Endocrinology
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Figure 1: EPO-associated reduction in blood glucose levels
(A). Blood glucose was measured in 3 months old tg6 mice and in their wt littermates. These
measurements were under non-fasting conditions. (B-E) Murine models were injected with rHuEPO
(180 U) or with diluent (control), 3 times weekly on alternating days, for a period of 2 weeks.
Glucose measurements were performed before EPO injections (initial), and one and two weeks
following EPO injections. These measurements were performed after 4 h of fasting. White and black
squares represent diluent and EPO injected mice respectively. (B). ob/ob mice, 8 weeks old. (C).
PTP1B-/- mice, 3 months old. (D). C57BL/6 mice, 3 months old. (E). BALB/c mice, 8 weeks old. The
number of mice in each group is indicated. Values depict mean ± SEM. *P < 0.02, **P < 0.003, ***
P < 0.0001.
131x117mm (600 x 600 DPI)
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Figure 2: Improved glucose tolerance conferred by EPO
GTTs were performed in the following mouse strains: (A). tg6 and wt littermates (solid and open
squares, respectively). (B). ob/ob. (C). PTP1B-/-. (D). C57BL/6. (E). BALB/c. For the rHuEPO and
diluent treated mice, GTTs were performed after 2 weeks of rHuEPO or diluent injections. The
number of mice in each group is indicated, solid and open squares indicate EPO and diluent injected
mice respectively. Values depict mean ± SEM. * P < 0.03, ** P < 0.003.
136x123mm (600 x 600 DPI)
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Manuscript submitted for review to Journal of Endocrinology
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Figure 3: Lower insulin levels (A) and increased sensitivity to insulin (B) in tg6 mice
(A). Serum concentrations of insulin in fed tg6 mice and their wt littermates, n = 5 mice/group. *P
= 0.04 (B) ITT was performed in tg6 mice and their wt littermates. Blood glucose values are
expressed as a percentage of glucose concentration, prior to insulin administration. n = 4
mice/group. Values depict mean ± SEM. *P = 0.05.
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45x14mm (600 x 600 DPI)
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Figure 4: EPO-associated reduction in hemoglobin A1c
Groups of ob/ob mice were injected with rHuEPO or with diluent (control), three times weekly on
alternating days for 3 weeks. Hemoglobin A1c was measured in the sera of the ob/ob mice (n = 5
and 6 for rHuEPO-treated and diluent-treated mice, respectively) and in the tg6 mice and their wt
littermates (n = 4 and 3 tg6 and wt mice, respectively) using ELISA. Values depict mean ± SEM. *P
< 0.03, **P = 0.001.
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Figure 5 : Reduced weight despite higher food consumption in tg6 mice
Body weight and food consumption of tg6 mice (4 months old), as compared to their wt littermates.
(A) Body weight of female tg6 mice and their wt littermates (n = 17 (tg6) and 19 (wt) mice/group)
and of male tg6 mice and their wt littermates (n = 8 (tg6) and 6 (wt) mice/group), *P = 0.02, **P
< 0.004. (B) Weekly food consumption of female and male tg6 mice and their wt littermates, as
calculated over a period of 4 weeks. Results are expressed as grams of food/mouse/week. n = 5
mice/group. Values depict mean weekly consumption of food per mouse ± SEM. **P < 0.004
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Figure 6: tg6 mice have less epididymal fat tissue
(A). Spleens, livers, muscle (gastrocnemius) and epididymal fat were dissected from female tg6
mice and their wt littermates and were weighed. Organ weights were normalized to total body
weight. n = 3 mice/group, values depict mean ± SEM, *P = 0.05, **P = 0.014. (B). Photographs of
mouse cavities; the arrows indicate the epididymal fat.
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Figure 7: EPO administration attenuates weight gain in ob/ob mice
Weight gain (A) and food consumption (B) of ob/ob mice injected with EPO or with diluent (control).
Body weight is presented as percentage change from initial body weight. Food consumption of
individual ob/ob mice was measured for a period of 2 weeks. Results are expressed as grams of
food/mouse/week. n = 6 mice/group. Values depict mean ± SEM. * P = 0.03, ** P = 0.003.
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