-mice that die in utero due to disruption of definitive erythropoiesis8
are rescued by the erythroid expressing Tg
-mice) that restores erythropoiesis. Although Tg
-mice show no gross developmental or morphological defect, we show here that they exhibit abnormally increased weight gain from the first week after birth. By10 months, the body weight of Tg
-mice exceeded that of WT-mice by 60% in females and 30% in males. The greater accelerated weight gain in female Tg
-mice may be related to estrogen-dependent Epo production that has been implicated in estrus cycle-dependent uterine angiogenesis23
The excessive body weight in Tg-mice was due to increased white fat mass since neither brown fat nor lean mass were altered, suggesting an obese phenotype. Accompanying the disproportionate white fat accumulation was deterioration of insulin sensitivity, evident by 4 months in female mice and 6 months in male mice. These results link Epo activity in non-hematopoietic tissues to white fat mass accumulation and propose an effect of endogenous Epo in maintaining normal fat mass. Hematocrit and weight gain in EpoR+/−-mice with or without the TgEpoRE transgene were indistinguishable from WT-mice providing evidence that the increase of weight gain in Tg-mice is a consequence of loss of EpoR expression in non-hematopoietic tissue rather than transgene-related. Red cell production appeared normal in Tg-mice and Epo treatment in both WT- and Tg-mice increased hematocrit. However, only WT-mice exhibited the decreased fat mass compared with saline-treated control mice. In ob/ob-mice with Epo treatment, phlebotomy prevented the increase of hematocrit but did not affect the Epo-induced decrease of body weight gain in these mice. These data demonstrate that the regulatory effect of Epo on fat mass accumulation is independent of erythropoiesis.
Decreased total activity and rate of oxygen consumption in Tg
-mice was detected at 2 months, before the massive increase in fat mass. In contrast, Epo treatment in HFD-fed WT-mice reduced fat mass accumulation, increased total activity and reduced food intake. These changes as consequences of alteration in Epo signaling indicate a breakdown of energy homeostasis leading to disruption of stability in body fat storage. Rodents and humans exposed to high altitude exhibit decreased food intake, loss of body fat with accompanying decreased plasma glucose and insulin24,25
. It is tempting to speculate that hypoxia inducible Epo may contribute to hypophagia and loss of body fat associated with high altitude.
The hypothalamus is an organ responsible for regulating energy metabolism. Two populations of neurons in the arcuate nucleus of hypothalamus act in an opposing manner to control of food intake, energy expenditure and glucose metabolism. POMC-expressing neurons sense the change in peripheral regulatory hormones, leptin and insulin, and respond by secreting α-MSH that stimulates melanocortin 3 and 4 receptors in target neurons to decrease food intake and increase energy expenditure26,27
. Activation of NPY/ARGP neurons expressing orexigenic neuron peptides NPY and AGRP stimulates orexigens MCH and orexins expression in lateral hypothalamus target neurons to increase food intake and decrease energy expenditure26-28
. Surprisingly, we observed high level of EpoR expression in murine hypothalamus localized to POMC neurons, and Epo regulated POMC gene expression but not anabolic hypothalamic neuropeptide gene expression. Furthermore, decreased POMC expression due to lack Epo signaling in Tg
-mice hypothalamus is consistent with decreased energy expenditure, increased metabolic efficiency and development of obesity. Increased POMC expression induced by Epo treatment provides an explanation for the decreased food intake, increased energy expenditure and decreased metabolic efficiency. These results show that Epo regulates energy homeostasis at least through its target, POMC neurons, in the hypothalamus. However, Tg-
mice with 50% POMC expression are not hyperphagic and the development of obesity is more gradual than that in Pomc−/−
mice reflecting a POMC-dose-dependent effect29
. Recent observations of weight gain regulation and high fat diet induced leptin resistance by PPARγ in the CNS30,31
suggest additional possible mechanisms for Epo action, given the effect of Epo on PPARγ in WAT and in differentiating preadipocytes. However, PPARγ activity in the rat hypothalamus localized to the paraventricular nucleus31
and not in the arcuate nucleus where we localize EpoR expression in POMC neurons.
The high level of EpoR expression in WT-WAT and the obese phenotype in Tg-mice that lack EpoR expression in WAT led us to explore Epo activities during preadipocyte proliferation and differentiation. Epo binding to the EpoR homodimer in erythroid progenitor cells activates JAK2 and downstream ERK and p38MAPK signaling pathways5-7,32
. Similarly, Epo activates ERK and p38MAPK in differentiating preadipocytes, consistent with reports of ERK activity increasing PPARγ phosphorylation and decreasing PPARγ activity19,33
. Adipocyte number is largely established during childhood and adolescence34
. A recent study also suggests that early adipocyte progenitor cell number plays a critical role in fat mass determination35
. Epo treatment during preadipocyte differentiation decreased the terminal expansion of cell number following MDI induction. Expansion of growth-arrested 3T3-L1 cells during differentiation induction is a critical step for subsequent adipogenic gene expression and adipocyte phenotype decision36
. The effect of Epo in reducing cell number of differentiating preadipocyte cultures suggests that loss of Epo activity in WAT may result in an increase of total adipocyte number. Indeed, Tg
-WAT exhibits marked increase in fat cell number and decrease in fat cell size despite the disproportionate increased fat mass accumulation. Mice homozygous for a PPARγ-2 phosphorylation site mutation (S112A) show a WAT phenotype of greater number and smaller size of adipocytes compared with WT-WAT37
, indicating that reduced or absent phosphorylation of PPARγ-2 can lead to increased fat cell number in vivo36
. Similarly, endogenous Epo signaling in WAT may contribute to regulation of adipocyte number.
Multiple hormones including insulin, leptin and adiponectin regulate food intake and energy expenditure through the CNS17,21
. Plasma insulin was significantly elevated in Tg
-mice when their obese phenotype became noticeable, suggesting that insulin resistance may be a consequence of obesity in Tg
-mice. Serum leptin and adiponectin levels appeared proportional to fat content - elevated with increased fat mass in Tg
-mice, and reduced with decreased fat mass in Epo treated WT-mice. Both leptin and adiponectin mRNA level in WAT are comparable between WT- and Tg
-mice. Epo treatment in WT-MEF cell cultures shows no effect in regulation of leptin and adiponectin gene expression both before and after differentiation. In addition, Epo treatment significantly reduced fat mass in leptin deficient ob/ob
-mice, indicating that leptin signaling may contribute but is not required for Epo regulation of body weight. Adiponectin is implicated in regulation of gluconeogenesis, fatty acid oxidation, food intake and energy expenditure by stimulating AMP-activated protein kinase and peroxisome proliferator-activated receptor-α18,38
, and declines in obese patients and animal models that are insulin resistant, suggesting that decreased adiponectin level contributes to development of obesity and insulin resistance39
. However, serum adiponectin levels in young Tg
-mice with modest obesity prior to onset of insulin resistance were higher than those in control WT-mice. Hence, adiponectin is an unlikely causal factor of obesity and insulin resistance in Tg
-mice. However, EpoR expression is elevated in WAT from obese WT-mice and ob/ob
-mice and we observed an increase expression of proinflammatory adipokines in Tg
- and ob/ob
-WAT and reduced IL-10 in Tg
-WAT. The improved insulin resistance with Epo treatment in ob/ob
-mice and WT-mice on HFD may relate to the immune-modulatory effects associated with Epo40
in non-hematopoietic tissue that is absent in Tg
Reports of Epo stimulated activation of an EpoR interaction with the GM-CSF/IL-3/IL-5 receptor common β-chain in hematopoietic and non-hematopoietic cells or tissues raised the possibility of an alternate Epo receptor41-43
. The studies presented here do not directly address the nature of the non-hematopoietic Epo receptor. However, the relatively high level of EpoR expression that we observed in the hypothalamus and WAT are consistent with the hypothesis that EpoR expression level determines Epo response and formation of the “classical” EpoR in these non-hematopoietic tissues.
While the essential role of Epo in erythropoiesis has been well established, the biological activity of Epo in non-hematopoietic tissues remains poorly understood. In the current study, we found significant EpoR expression in WAT and hypothalamus. Epo regulates POMC expression in hypothalamus and ablation of Epo signaling in hypothalamus disrupts energy homeostasis and leads to development of obesity and insulin resistance in Tg-mice. The effects of Epo in suppressing preadipocyte differentiation in vitro are associated with increased PPARγ phosphorylation and reduced preadipocyte expansion during differentiation. The lack of Epo signaling in WAT results in an increase in adipocyte number, which may lead to predisposition to this central defect-driven obesity. Together, these data indicate a role of Epo in maintaining normal fat mass in mice and suggest that further investigations are warranted to fully understand the central regulation of Epo in energy homeostasis.
-mice containing the Tg
transgene (GATA-1 locus hematopoietic regulatory domain driving mouse EpoR cDNA12
) were established on the EpoR−/−
(C57BL/6) background (Jackson Laboratories, Bar Harbor, ME, USA) to obtain EpoR−/−Tg
-mice. Heterozygous EpoR+/−
-mice and EpoR+/−Tg
-mice with the transgene were generated. Genotypes were identified by PCR (Neo gene (339bp), wild type EpoR (387bp) and transgene (303bp)) (Supplementary Table S1
Mice including WT-mice (C57BL/6), ob/ob
-mice (Jackson Laboratories) and lean littermate control-mice were maintained on a 12-h light/dark cycles and fed regular chow NIH-07 diet (Zeigler Brothers, Inc., Gardnerds, PA). Animal procedures following National Institutes of Health guidelines were approved by NIDDK Animal Care and Use Committee. The mouse strains and the number, gender and age are listed in Supplementary Table S2
Body weight and composition
Body weight was measured using the same balance by the same person. Body composition was measured using the EchoMRI 3-in-1™ (Echo Medical Systems).
Computed tomography for fat determination
X-ray computed tomography (MicroCat II, Imtek, Inc) was used for imaging rodents at 50-100 microns resolutions under anesthesia. Three-dimensional reconstructions of microCT images utilized the Hamming algorithm for soft tissue and semi-automatic algorithms to identify fat by anatomic sites (visceral, subcutaneous and nuchal-scapular brown fat) with the established Hounsfield Units. Amira 4.1 software for three-dimensional image analysis (Mercury ComputerSystems, Chelmsford, MA) was used to determine the total volume for each fat type.
Epo treatment with hematocrit measurement
For Epo treatment (3,000 Units/Kg; Epoetin alpha, Amgen Manufacturing, Thousand Oaks, CA), mice received intraperitoneal injection three times/week. Hematocrit was measured manually before and every week after Epo application.
Serum parameter analyses
Triglycerides were determined using Infinity™ Triglycerides reagents (Thermo DMA, Louisville, CO). Insulin, leptin and adiponectin concentrations were determined by radioimmunoassay (Linco Research Inc. St. Charles, MO).
Glucose Tolerance Test and Insulin Tolerance Test
Mice fasted overnight were injected intraperitoneally with glucose (2.0g/kg body weight) or insulin (Humulin R, 0.75 mU/kg). Blood glucose levels were measured before (0 min) and up to 120 min after the injection (3 μl from tail vein; Elite Glucometer (Bayer, Elkhart, IN)). For determination of insulin secretion in response to glucose stimulation, 30 μl of blood was collected before (0 min) and up to120 min after glucose injection; serum was prepared and frozen on dry ice immediately for later assessment.
Spontaneous locomotor and explorative activities were measured using an open field system (AccuScan Instruments, 8”× 8” configuration). Mice are tested between 6 am and 11 am for 3 consecutive days. Data were recorded for 60 min after 10 min acclimation to new environment and are expressed as averages from 3 days of testing.
Indirect calorimetry and food intake
Indirect calorimetry including oxygen consumption and activity measurements were assessed 44
. Oxygen consumption and CO2
production were measured at 24C° every 20 min for 47 hours using an eight-chamber Oxymax System (Columbus Instruments, OH; 2.5 l chambers with wire mesh floors and gauze as bedding, using 0.6 l/min flow rate, 90 sec purge, and 30 sec measure; one mouse/chamber). The system was calibrated using a defined mixture of O2
. Mice were allowed to adapt to individual housing for three weeks. During testing food and water were provided ad libitum
. Motor activities were determined by infrared beam interruption (Opto-Varimex mini; Columbus). Resting metabolic rate was calculated with ambulation equal zero. The respiratory exchange ratio (RER) was calculated as the ratio between VCO2
. Data were expressed as averages of 46 hours excluding the first hour of the experiment. Oxygen consumption data were normalized to (body weight)0.75
Total RNA extracted from cells or tissues using TRIzol (Invitrogen) was treated with Turbo DNase (Ambion) and 2 μg was reverse transcribed (MultiScribe Reverse Transcriptase (ABI)) for quantitative PCR assays. Relative mRNA quantification of adipokine genes used Taq
man gene expression assays (Supplementary Table S3
) (Invitrogen). For absolute quantification of mouse EpoR mRNA in fat and hypothalamus, probe-based Taqman PCR and mouse EpoR and S16 cDNA plasmid were used (16S, internal control). For relative mRNA quantification of all other genes, SYBR green real-time RT-PCR was used with normalization to house keeping genes β-actin and 18S using the Delta-Delta CT method. Primers and probes used in Taqman PCR and SYBR green real-time RT-PCR are listed in Supplementary Table S4
Preparation of primary neonatal cardiomyocytes
Hearts from postnatal 1 day mice were finely minced. Tissue was digested with collagenase/pancreatin at 37°C for 10 min, disrupted by gently pipetting (10X), and the supernatant was transferred into cold PBS with 50% FBS. After three cycles of digestion, cells were plated in cardiomyocyte culture medium SmGM®-2 medium (CC3181 and CC4149, Lonza), non-adherent cells (cardiomyocytes) were collected after 40 min. and replated into collagen I coated dishes.
Fractionation of cells from white adipose tissue
Inguinal fat pads were harvested, digested with 1mg/ml collagenase (type II) (37°C, 50 min), and reacion stopped with equal volume of 50% FBS in DMEM. After centrifugation (600g, 10 min), floating mature adipocytes were separated from the pellet of stromal vascular (SV) cells. Cells were plated and analyzed after 24 hours.
Measurement of fat cell size
Cell-size distribution was measured in gonadal fat pad from Tg
- and WT-mice using Beckman Coulter Multisizer III45
. Tissue samples from multiple areas and critically the same locations in each fat pad were immediately fixed in osmium tetroxide (37°C, 48 h), and adipose cell size was determined with a 400 μ
m aperture (20-240 μ
m cell size range). 6,000 events were counted for each sample.
Preparation of MEF cells
Embryos at day 14p.c. (brain and dark red organs removed) were finely minced and digested with trypsin-EDTA (Gibco 25300) (37°C) at 1ml per embryo with gentle shaking for 30 min (gentle disruption pipetting up and down 10X every 10 min), and reaction stopped with equal volume of cold PBS with 50% FCS. The solution was filtered (falcom 40mm cell strainer) and cells were collected by centrifugation, washed twice with fresh MEF culture medium (DMEM-high glucose, 10% FBS, 1% pen/strep, Gibco), suspended in warm MEF culture medium and plated (1 embryo per dish). Medium was changed at 1 hour to remove the unattached cells. Remaining cells were cultured and then frozen for later use.
MEF cells and 3T3-L1 cells (ATCC, Manassas, VA) were grown in 10 % CO2, in (DMEM, 10% FBS, 1% pen/strep, Gibco). For preadipocyte differentiation, cells at 100% confluency were stimulated in medium DMEM medium supplemented with 0.5mM IBMX, 5ug/ml insulin and 1uM dexamethasone (Sigma) in the absence or presence of Epo. After 3 days, maintenance medium (DMEM, 10% FBS, 1% pen/strep, 5ug/ml insulin) was used and changed every day. Fresh Epo was added at medium changes.
Oil red O staining
After preadipocyte differentiation, cells were washed with PBS gently and’ fixed (10% formalin, 1 hour, room temperature). Formalin was removed and fresh oil red O solution (0.3% in 60% isopropanol) was added to stain the cells (1 hour, gentle swirling). Cells were rinsed with distilled water thoroughly. For quantification, stained dishes were dried, oil red O was resuspended in isopropanol (1 min) and solution OD was determined at 510nm.
WAT and MEF cells were homogenized, incubated in lysis buffer (50 mM Tris-HCI buffer, pH 7.4, 1% NP40, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) with phosphotase and proteinase inhibitors (Roche) (30 min on ice), and cell debris was removed by centrifugation. Protein concentration was determined using Pierce BCA protein assay kit (Thermo Scientific). Sample proteins (30μg/each) were electrophoretically separated, blotted using XCell SureLock
™ Mini-Cell system (Invitrogen)(supplemental information
) and visualized using protein specific antibodies (Supplementary Table S5
) and the Amersham ECL™ Advance Western Blotting Detection System (GE Healthcare Bio-Sciences Corp). Quantitative analysis was performed by measuring integrated density with NIH image J system and normalized with β-actin.
After dewaxing and rehydration, antigen retrieval was performed by autoclaving using antigen unmasking solution (Vector Laboratories). Rabbit anti-mouse POMC antibody (kindly provided by Dr. Peng Loh; Cellular Neurobiology Section, NICHD/NIH) and FITC-conjugated goat anti-rabbit IgG (M30201, Invitrogen) as a secondary antibody were used for labeling POMC neurons. Concerns about commercial EpoR antibodies suggest caution regarding their use46,47
. Since we found variability among commercial lots of EpoR polyclonal antibody, we used the monoclonal antibody developed at Genetics Institute against the EpoR extracellular domain (mh2er/16.5.1) that previously provided consistent results for immunohistochemistry48,49
and Alexa594-conjugated goat anti-mouse IgG (A11020, Invitrogen) in detecting mEpoR.
Preparation of primary adult hypothalamic neurons
Adult neurons were isolated from hypothalamus50
and cultured for 24 hours, then treated with Epo (5U/ml) or saline for 24 hours. Total RNA were extracted and analyzed for POMC, NPY, AGRP, prepro-orexin and pro-MCH gene expression.
The data are expressed as means ± SEM. Comparisons between two groups were made using two-tailed nonpaired Student’s t-test. Statistical differences between three or more groups were evaluated by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison post hoc tests at α=0.05. A p value of less than 0.05 was regarded to be statistically significant.