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Erythropoietin regulates metabolic response in mice via receptor expression in adipose tissue, brain, and bone

  • Constance Tom Noguchi
    Correspondence
    Offprint requests to: Constance Tom Noguchi, Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 9N319, 10 Center Drive, MSC-1822, Bethesda, MD 20892–1822
    Affiliations
    Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
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Open ArchivePublished:September 17, 2020DOI:https://doi.org/10.1016/j.exphem.2020.09.190

      Highlights

      • Mouse models demonstrate erythropoietin metabolic response mediated by erythropoietin receptor expression in adipose tissue, brain, and bone.
      • Erythropoietin regulation of fat mass in white adipose tissue is gender dependent.
      • Cerebral erythropoietin regulation of fat mass and hypothalamus inflammation during feeding of a high-fat diet is gender dependent.
      • Erythropoietin regulates adipogenesis and osteogenesis in bone.
      • Bone loss accompanying erythropoietin-stimulated erythropoiesis is mediated by a direct osteoblast response.
      Erythropoietin (EPO) acts by binding to erythroid progenitor cells to regulate red blood cell production. While EPO receptor (Epor) expression is highest on erythroid tissue, animal models exhibit EPO activity in nonhematopoietic tissues, mediated, in part, by tissue-specific Epor expression. This review describes the metabolic response in mice to endogenous EPO and EPO treatment associated with glucose metabolism, fat mass accumulation, and inflammation in white adipose tissue and brain during diet-induced obesity and with bone marrow fat and bone remodeling. During high-fat diet-induced obesity, EPO treatment improves glucose tolerance, decreases fat mass accumulation, and shifts white adipose tissue from a pro-inflammatory to an anti-inflammatory state. Fat mass regulation by EPO is sex dimorphic, apparent in males and abrogated by estrogen in females. Cerebral EPO also regulates fat mass and hypothalamus inflammation associated with diet-induced obesity in males and ovariectomized female mice. In bone, EPO contributes to the balance between adipogenesis and osteogenesis in both male and female mice. EPO treatment promotes bone loss mediated via Epor in osteoblasts and reduces bone marrow adipocytes before and independent of change in white adipose tissue fat mass. EPO regulation of bone loss and fat mass is independent of EPO-stimulated erythropoiesis. EPO nonhematopoietic tissue response may relate to the long-term consequences of EPO treatment of anemia in chronic kidney disease and to the alternative treatment of oral hypoxia-inducible factor prolyl hydroxylase inhibitors that increase endogenous EPO production.

      Graphical Abstract

      Erythropoietin (EPO), produced in the kidney, is the primary regulator of erythropoiesis [
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      Now a Nobel gas: oxygen.
      ]. EPO is regulated by hypoxia [
      • Pugh CW
      • Ratcliffe PJ
      New horizons in hypoxia signaling pathways.
      ]. Hypoxia-inducible factor (HIF) heterodimer (ARNT/HIF-α; primarily HIF-2α for EPO) induces EPO by binding to the EPO gene hypoxia-responsive element [
      • Semenza GL
      Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis.
      ,
      • Wang GL
      • Jiang BH
      • Rue EA
      • Semenza GL
      Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.
      ,
      • Rankin EB
      • Biju MP
      • Liu Q
      • et al.
      Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin in vivo.
      ]. HIF-α is stable and active under hypoxia and is targeted at normoxia by oxygen-dependent prolyl hydroxylase-domain (PHD) enzymes and factor-inhibiting HIF-1 [
      • Jaakkola P
      • Mole DR
      • Tian YM
      • et al.
      Targeting of HIF-alpha to the von Hippel–Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.
      ,
      • Mahon PC
      • Hirota K
      • Semenza GL
      FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity.
      ]. Proline hydroxylation by PHD2 targets HIF-α for ubiquitination by Von Hippel–Lindau protein and proteasome degradation [
      • Jaakkola P
      • Mole DR
      • Tian YM
      • et al.
      Targeting of HIF-alpha to the von Hippel–Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.
      ,
      • Ohh M
      • Park CW
      • Ivan M
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      Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel–Lindau protein.
      ,
      • Minamishima YA
      • Moslehi J
      • Bardeesy N
      • Cullen D
      • Bronson RT
      • Kaelin Jr, WG
      Somatic inactivation of the PHD2 prolyl hydroxylase causes polycythemia and congestive heart failure.
      ]. Mutations in genes for PHD2, VHL, and HIF2A, as well as EPO and EPO receptor (Epor), contribute to congenital erythrocytosis [
      • Lappin TR
      • Lee FS
      Update on mutations in the HIF: EPO pathway and their role in erythrocytosis.
      ] and suggest alternate modalities to stimulate erythropoiesis. Recently, HIF-prolyl hydroxylase inhibitors, small molecule oral agents that stimulate production of endogenous erythropoietin, have been approved in China and Japan for treatment of anemia associated with chronic kidney disease [
      • Chen N
      • Hao C
      • Liu BC
      • et al.
      Roxadustat treatment for anemia in patients undergoing long-term dialysis.
      ,
      • Chen N
      • Hao C
      • Peng X
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      Roxadustat for anemia in patients with kidney disease not receiving dialysis.
      ], although adverse events with long-term administration remain unknown [
      • Sanghani NS
      • Haase VH
      Hypoxia-inducible factor activators in renal anemia: Current clinical experience.
      ].
      Animal models suggest that EPO can promote a nonhematopoietic response mediated via Epor expression beyond erythroid tissue and include protection against ischemic stress and injury in brain, vascular endothelium, heart, and skeletal muscle [
      • Wang L
      • Di L
      • Noguchi CT
      Erythropoietin, a novel versatile player regulating energy metabolism beyond the erythroid system.
      ,
      • Suresh S
      • Rajvanshi PK
      • Noguchi CT
      The many facets of erythropoietin phsyiologic and metabolic response.
      ]. The nonhematopoietic EPO responses may also relate to EPO production by HIF-prolyl hydroxylase inhibitors. Reviewed here is the metabolic response to endogenous and exogenous EPO such as glucose tolerance, anti-inflammatory response in white adipose tissue (WAT) and brain, gender-specific fat mass regulation particularly during diet-induced obesity in mice, and adipogenic/osteogenic balance in bone maintenance [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Alnaeeli M
      • Raaka BM
      • Gavrilova O
      • Teng R
      • Chanturiya T
      • Noguchi CT
      Erythropoietin signaling: a novel regulator of white adipose tissue inflammation during diet-induced obesity.
      ,
      • Zhang Y
      • Rogers HM
      • Zhang X
      • Noguchi CT
      Sex difference in mouse metabolic response to erythropoietin.
      ,
      • Dey S
      • Cui Z
      • Gavrilova O
      • Zhang X
      • Gassmann M
      • Noguchi CT
      Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.
      ,
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ].

      EPO receptor and EPO-stimulated signaling beyond erythropoiesis

      EPO stimulates erythroid progenitor cell survival, proliferation, and differentiation regulating the production of two million erythrocytes per second in the human body. Mice that lack Epor die in utero of severe anemia [
      • Wu H
      • Liu X
      • Jaenisch R
      • Lodish HF
      Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.
      ,
      • Lin CS
      • Lim SK
      • D'Agati V
      • Costantini F
      Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis.
      ]. EPO binding to the cell surface Epor homodimer on erythroid progenitor cells activates Epor cytoplasmic domain associated JAK2 and phosphorylation of Epor, STAT, AKT, ERK, and other downstream signaling pathways [
      • Witthuhn BA
      • Quelle FW
      • Silvennoinen O
      • et al.
      JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.
      ,
      • Kuhrt D
      • Wojchowski DM
      Emerging EPO and EPO receptor regulators and signal transducers.
      ]. EPO binding to Epor induces erythroid transcription factors GATA1 and TAL1, which also transactivate Epor via a GATA-binding site and three TAL1-binding (E-boxes) motifs in the proximal promoter. Epor expression and EPO sensitivity is greatest on erythroid progenitor cells [
      • Zon LI
      • Youssoufian H
      • Mather C
      • Lodish HF
      • Orkin SH
      Activation of the erythropoietin receptor promoter by transcription factor GATA-1.
      ,
      • Kassouf MT
      • Hughes JR
      • Taylor S
      • et al.
      Genome-wide identification of TAL1′s functional targets: insights into its mechanisms of action in primary erythroid cells.
      ,
      • Rogers H
      • Wang L
      • Yu X
      • et al.
      T-cell acute leukemia 1 (TAL1) regulation of erythropoietin receptor and association with excessive erythrocytosis.
      ,
      • Broudy VC
      • Lin N
      • Brice M
      • Nakamoto B
      • Papayannopoulou T
      Erythropoietin receptor characteristics on primary human erythroid cells.
      ].
      The GATA motif and E-boxes also provide for Epor expression in select nonhematopoietic tissues including the endothelial/cardiovascular system, brain, skeletal muscle, fat depots, and bone [
      • Wang L
      • Di L
      • Noguchi CT
      Erythropoietin, a novel versatile player regulating energy metabolism beyond the erythroid system.
      ,
      • Suresh S
      • Rajvanshi PK
      • Noguchi CT
      The many facets of erythropoietin phsyiologic and metabolic response.
      ], and can be transactivated in part by other GATA proteins including GATA2, GATA3, and GATA4 [
      • Ogilvie M
      • Yu X
      • Nicolas-Metral V
      • et al.
      Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts.
      ,
      • Yu X
      • Shacka JJ
      • Eells JB
      • et al.
      Erythropoietin receptor signalling is required for normal brain development.
      ,
      • Wang L
      • Jia Y
      • Rogers H
      • Wu YP
      • Huang S
      • Noguchi CT
      GATA-binding protein 4 (GATA-4) and T-cell acute leukemia 1 (TAL1) regulate myogenic differentiation and erythropoietin response via cross-talk with Sirtuin1 (Sirt1).
      ,
      • Gaine ME
      • Sharpe DJ
      • Smith JS
      • et al.
      GATA2 regulates the erythropoietin receptor in t(12;21) ALL.
      ]. Endothelial cells expressing Epor induced by reduced oxygen and/or nitric oxide [
      • Beleslin-Cokic BB
      • Cokic VP
      • Yu X
      • Weksler BB
      • Schechter AN
      • Noguchi CT
      Erythropoietin and hypoxia stimulate erythropoietin receptor and nitric oxide production by endothelial cells.
      ,
      • Beleslin-Cokic BB
      • Cokic VP
      • Wang L
      • et al.
      Erythropoietin and hypoxia increase erythropoietin receptor and nitric oxide levels in lung microvascular endothelial cells.
      ,
      • Cokic BB
      • Cokic VP
      • Suresh S
      • Wirt S
      • Noguchi CT
      Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.
      ] exhibit EPO proliferative and chemotactic response [
      • Anagnostou A
      • Lee ES
      • Kessimian N
      • Levinson R
      • Steiner M
      Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells.
      ,
      • Anagnostou A
      • Liu Z
      • Steiner M
      • et al.
      Erythropoietin receptor mRNA expression in human endothelial cells.
      ]. In mice, Epor is required for vessel network development and for EPO-stimulated, endothelial nitric oxide synthase (eNOS)-mediated cardioprotection [
      • Kertesz N
      • Wu J
      • Chen TH
      • Sucov HM
      • Wu H
      The role of erythropoietin in regulating angiogenesis.
      ,
      • Teng R
      • Calvert JW
      • Sibmooh N
      • et al.
      Acute erythropoietin cardioprotection is mediated by endothelial response.
      ,
      • Van Der Meer P
      • Lipsic E
      • Henning RH
      • et al.
      Erythropoietin induces neovascularization and improves cardiac function in rats with heart failure after myocardial infarction.
      ,
      • Mihov D
      • Bogdanov N
      • Grenacher B
      • et al.
      Erythropoietin protects from reperfusion-induced myocardial injury by enhancing coronary endothelial nitric oxide production.
      ]. In neural cells, Epor is transactivated by GATA3, which is critical for morphological development of the nervous system [
      • Yu X
      • Shacka JJ
      • Eells JB
      • et al.
      Erythropoietin receptor signalling is required for normal brain development.
      ,
      • Pandolfi PP
      • Roth ME
      • Karis A
      • et al.
      Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.
      ]. In rodents, endogenous EPO contributes to maintenance and proliferation of neural progenitor cells and neuroprotection [
      • Yu X
      • Shacka JJ
      • Eells JB
      • et al.
      Erythropoietin receptor signalling is required for normal brain development.
      ,
      • Liu C
      • Shen K
      • Liu Z
      • Noguchi CT
      Regulated human erythropoietin receptor expression in mouse brain.
      ,
      • Tsai PT
      • Ohab JJ
      • Kertesz N
      • et al.
      A critical role of erythropoietin receptor in neurogenesis and post-stroke recovery.
      ,
      • Chen ZY
      • Asavaritikrai P
      • Prchal JT
      • Noguchi CT
      Endogenous erythropoietin signaling is required for normal neural progenitor cell proliferation.
      ], and exogenous EPO is neuroprotective for brain ischemia and injury [
      • Yu X
      • Shacka JJ
      • Eells JB
      • et al.
      Erythropoietin receptor signalling is required for normal brain development.
      ,
      • Sakanaka M
      • Wen TC
      • Matsuda S
      • et al.
      In vivo evidence that erythropoietin protects neurons from ischemic damage.
      ,
      • Bernaudin M
      • Marti HH
      • Roussel S
      • et al.
      A potential role for erythropoietin in focal permanent cerebral ischemia in mice.
      ]. In skeletal muscle myoblasts, Epor induced by GATA3, GATA4, and TAL1 and E-box-binding muscle regulator transcription factors MyoD and Myf5 [
      • Ogilvie M
      • Yu X
      • Nicolas-Metral V
      • et al.
      Erythropoietin stimulates proliferation and interferes with differentiation of myoblasts.
      ,
      • Wang L
      • Jia Y
      • Rogers H
      • Wu YP
      • Huang S
      • Noguchi CT
      GATA-binding protein 4 (GATA-4) and T-cell acute leukemia 1 (TAL1) regulate myogenic differentiation and erythropoietin response via cross-talk with Sirtuin1 (Sirt1).
      ] promotes transplanted myoblast survival and restored dystrophin expression in mdx mice [
      • Jia Y
      • Suzuki N
      • Yamamoto M
      • Gassmann M
      • Noguchi CT
      Endogenous erythropoietin signaling facilitates skeletal muscle repair and recovery following pharmacologically induced damage.
      ,
      • Jia Y
      • Warin R
      • Yu X
      • Epstein R
      • Noguchi CT
      Erythropoietin signaling promotes transplanted progenitor cell survival.
      ]. Endogenous EPO and exogenous EPO contribute to skeletal muscle repair in mice [
      • Jia Y
      • Suzuki N
      • Yamamoto M
      • Gassmann M
      • Noguchi CT
      Endogenous erythropoietin signaling facilitates skeletal muscle repair and recovery following pharmacologically induced damage.
      ,
      • Kuang S
      • Kuroda K
      • Le Grand F
      • Rudnicki MA
      Asymmetric self-renewal and commitment of satellite stem cells in muscle.
      ].

      EPO activity in nonhematopoietic tissue and regulation of fat mass

      Epor knockout mice can be rescued from death in utero by an erythroid-specific Epor transgene driven by GATA1 erythroid transcription regulatory regions, resulting in mice with erythroid-restricted Epor (ΔEPORE) [
      • Suzuki N
      • Ohneda O
      • Takahashi S
      • et al.
      Erythroid-specific expression of the erythropoietin receptor rescued its null mutant mice from lethality.
      ]. ΔEPORE mice have no gross morphological defects, indicating that nonhematopoietic Epor expression is not required for life [
      • Suzuki N
      • Ohneda O
      • Takahashi S
      • et al.
      Erythroid-specific expression of the erythropoietin receptor rescued its null mutant mice from lethality.
      ]. Although food intake is comparable between ΔEPORE and wild-type mice on a C57BL/6 background, ΔEPORE mice are glucose intolerant and become obese and insulin resistant with decreased metabolic rate and locomotor activity (Figure 1) [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ]. By age 8 months, female ΔEPORE mice exhibit a 150% increase in fat mass and a 55% increase in body weight, and male ΔEPORE mice exhibit a 40% increase in fat mass and a 20% increase in body weight compared with wild-type mice [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ]. Despite increases in fat mass, adipocyte size distribution in ΔEPORE gonadal fat pads shifted to smaller cell size [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ], indicating a disproportionate increase in adipocyte number with loss of nonhematopoietic Epor.
      Figure 1
      Figure 1Erythropoietin contributes to fat mass regulation. ΔEPOREmice with Epor restricted to erythroid tissue exhibit accelerated body weight gain because of increased fat mass and become obese. Conversely, C57BL/6 male mice fed a high-fat diet become obese, while EPO treatment concomitant with a high-fat diet increases hematocrit and protects against diet-induced obesity. In female mice, estrogen provides protection against diet-induced obesity, and EPO treatment increases hematocrit without change in fat mass. Ovariectomy in female mice abrogates the estrogen anti-obesity activity, and ovariectomized mice fed a high-fat diet become obese. Ovariectomized mice on a high-fat diet concomitant with EPO treatment exhibit an increased hematocrit and protection against diet-induced obesity.
      In wild-type mice, Epor expression in white adipose tissue is 60% the level of erythroid tissue (spleen) and, in brown adipose tissue, is an order of magnitude lower [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ]. Mice (C57BL/6 background) with adipocyte deletion of Epor also exhibited increase fat mass accumulation, insulin resistance, and reduced oxygen consumption and activity [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ]. These mice exhibit an increase of 20% in body weight because of increased fat mass by 30 weeks, compared with littermate control mice, and increased susceptibility to high-fat diet-induced obesity, glucose intolerance, and insulin resistance. Insulin activation of the serine/threonine kinase AKT (also known as protein kinase B) in adipocytes is required to stimulate glucose transporter 4 translocation to the membrane to increase glucose uptake [
      • Kearney AL
      • Cooke KC
      • Norris DM
      • et al.
      Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.
      ]. In erythroid cells EPO stimulates AKT signaling to promote survival, proliferation, and differentiation downstream of EPOR activation [
      • Ghaffari S
      • Kitidis C
      • Zhao W
      • et al.
      AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.
      ]. EPO treatment also activates AKT in adipocytes, but not in mice that lack Epor in adipocytes and also manifest reduced AKT phosphorylation compared with control mice [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ]. These mouse models illustrate that both endogenous and exogenous EPO activity contributes to regulation of fat mass and glucose homeostasis, in part via direct adipocyte EPO response to affect insulin signaling that may also be influenced by mouse background strain [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ,
      • Luk CT
      • Shi SY
      • Choi D
      • Cai EP
      • Schroer SA
      • Woo M
      In vivo knockdown of adipocyte erythropoietin receptor does not alter glucose or energy homeostasis.
      ].

      Exogenous EPO modulates body weight and fat mass accumulation

      Male mice treated with EPO exhibit increased hematocrit and decreased body weight when fed normal chow or reduced weight gain and fat mass accumulation when fed a high-fat diet (Figure 1) [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Katz O
      • Stuible M
      • Golishevski N
      • et al.
      Erythropoietin treatment leads to reduced blood glucose levels and body mass: insights from murine models.
      ]. Further evidence that elevated serum EPO increased hematocrit and decreased blood glucose and body weight is provided by mice treated with EPO and transgenic mice with constitutive high human EPO [
      • Katz O
      • Stuible M
      • Golishevski N
      • et al.
      Erythropoietin treatment leads to reduced blood glucose levels and body mass: insights from murine models.
      ]. Gene electrotransfer in skeletal muscle to increase EPO expression in obese mice also revealed increased erythropoiesis and reduced body weight and fat mass, improved glucose tolerance, and increased fat metabolism [
      • Hojman P
      • Brolin C
      • Gissel H
      • et al.
      Erythropoietin over-expression protects against diet-induced obesity in mice through increased fat oxidation in muscles.
      ]. In contrast, ΔEPORE mice with EPO receptor restricted to erythroid tissue and mice with targeted deletion of Epor in adipocytes exhibited no significant changes in fat mass/body weight with EPO-stimulated erythropoiesis [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ]. This indicates that exogenous EPO regulation of body weight/fat mass is independent of EPO-stimulated erythropoiesis and is mediated by EPO activity in nonhematopoietic tissue, especially in adipose tissue.
      EPO treatment during a high-fat-diet in mice increased metabolic activity and white adipose tissue cellular respiration capacity, fatty acid utilization, mitochondrial biogenesis and fatty acid oxidation-associated gene expression, metabolic regulator Pgc-1α, and cytochrome C protein compared with vehicle-treated and pair-fed diet-induced obese mice [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ]. Analogous changes were observed in EPO-treated mouse and human adipocyte cultures. In contrast, these activities and gene expression were reduced in white adipose tissue of mice with adipocyte deletion of Epor [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ]. EPO-associated response in cellular mitochondrial respiration and oxidative metabolism extend the role of EPO/Epor beyond regulation of erythropoiesis and oxygen transport capacity. Nonerythroid EPO activity contributes to increased energy expenditure in white adipose tissue and enhances the ability of adipocytes to metabolize fatty acid and to potentially protect against obesity.
      Brown adipose tissue with high mitochondria content maintains body temperature by release of chemical energy as heat via nonshivering thermogenesis [
      • Srivastava S
      • Veech RL
      Brown and brite: The fat soldiers in the anti-obesity fight.
      ]. The browning of white adipose tissue is characterized by increased levels of uncoupling protein UCP1, which uncouples electron transport from oxidative phosphorylation to generate heat [
      • Srivastava S
      • Veech RL
      Brown and brite: The fat soldiers in the anti-obesity fight.
      ,
      • Nedergaard J
      • Cannon B
      The browning of white adipose tissue: some burning issues.
      ]. Increasing beige adipocytes in white adipose tissue is of particular interest and has the potential to utilize energy-dissipating thermogenesis to reduce fat storage and promote a lean phenotype. EPO treatment in mice increased expression and protein of brown fat-associated genes including UCP1 in adipocytes from subcutaneous fat independent of change in body weight [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ]. Corresponding expression was decreased in mice with targeted deletion of Epor in adipocytes that was unchanged with EPO treatment. Primary adipocyte cultures also exhibit an analogous EPO-stimulated increase in brown fat-associated genes. Citrate synthase, the first enzyme in the tricarboxylic acid cycle, is an indicator of mitochondrial function. EPO treatment in mice increased citrate synthase activity in adipocytes from white adipose tissue but not from brown adipose tissue or from adipose tissue with adipocyte deletion of Epor. Hence, endogenous EPO and EPO administration contribute to white adipose tissue metabolism including direct adipocyte EPO response. In white adipose tissue, the nuclear receptor protein peroxisome proliferator-activated receptor (PPAR)-α reduced obesity-related inflammation and enhanced expression of brown fat-associated gene expression, including the thermogenesis effector UCP1 and transcription factor PRDM16 [
      • Tsuchida A
      • Yamauchi T
      • Takekawa S
      • et al.
      Peroxisome proliferator-activated receptor (PPAR)alpha activation increases adiponectin receptors and reduces obesity-related inflammation in adipose tissue: comparison of activation of PPARalpha, PPARgamma, and their combination.
      ,
      • Rachid TL
      • Silva-Veiga FM
      • Graus-Nunes F
      • Bringhenti I
      • Mandarim-de-Lacerda CA
      • Souza-Mello V
      Differential actions of PPAR-alpha and PPAR-beta/delta on beige adipocyte formation: A study in the subcutaneous white adipose tissue of obese male mice.
      ]. EPO stimulated an increase in PPAR-α in white adipose tissue in cooperation with SIRT1 activity, an NAD-dependent class III histone deacetylase sirtuin [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ]. EPO induced PPAR-α mediates the increases in brown fat-associated gene and mitochondrial gene expression, oxygen consumption rate, and fatty acid oxidation [
      • Wang L
      • Teng R
      • Di L
      • et al.
      PPARalpha and Sirt1 mediate erythropoietin action in increasing metabolic activity and browning of white adipocytes to protect against obesity and metabolic disorders.
      ].
      In brown fat of young male mice, EPO treatment increased PRDM16, which regulates brown adipocyte differentiation, UCP1 expression, STAT3 activation, and secretion of fibroblast growth factor 21 (FGF21), and improved glucose tolerance and insulin sensitivity [
      • Kodo K
      • Sugimoto S
      • Nakajima H
      • et al.
      Erythropoietin (EPO) ameliorates obesity and glucose homeostasis by promoting thermogenesis and endocrine function of classical brown adipose tissue (BAT) in diet-induced obese mice.
      ]. In liver, EPO regulated lipid metabolism, increased lipolysis, decreased lipogenesis, activated STAT3 signaling, and also increased FGF21 in a SIRT1-dependent manner [
      • Tsuma Y
      • Mori J
      • Ota T
      • et al.
      Erythropoietin and long-acting erythropoiesis stimulating agent ameliorate non-alcoholic fatty liver disease by increasing lipolysis and decreasing lipogenesis via EPOR/STAT pathway.
      ,
      • Hong T
      • Ge Z
      • Zhang B
      • Meng R
      • Zhu D
      • Bi Y
      Erythropoietin suppresses hepatic steatosis and obesity by inhibiting endoplasmic reticulum stress and upregulating fibroblast growth factor 21.
      ], suggesting that EPO can suppress obesity and hepatic steatosis. In obese male ob/ob mice, EPO treatment provided protection against obesity, reduced body weight, and hemoglobin A1c [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Katz O
      • Stuible M
      • Golishevski N
      • et al.
      Erythropoietin treatment leads to reduced blood glucose levels and body mass: insights from murine models.
      ]. EPO-stimulated metabolic response is dependent on EPO dose and duration of treatment [
      • Foskett A
      • Alnaeeli M
      • Wang L
      • Teng R
      • Noguchi CT
      The effects of erythropoietin dose titration during high-fat diet-induced obesity.
      ]. EPO induction at high altitude and the potential for EPO regulation of fat mass may contribute to the lower prevalence of obesity at high altitude [
      • Voss JD
      • Allison DB
      • Webber BJ
      • Otto JL
      • Clark LL
      Lower obesity rate during residence at high altitude among a military population with frequent migration: a quasi experimental model for investigating spatial causation.
      ,
      • Diaz-Gutierrez J
      • Martinez-Gonzalez MA
      • Pons Izquierdo JJ
      • Gonzalez-Muniesa P
      • Martinez JA
      • Bes-Rastrollo M
      Living at higher altitude and incidence of overweight/obesity: prospective analysis of the SUN cohort.
      ].

      Gender-specific response to EPO regulation of fat mass

      ΔEPORE mice with Epor restricted to erythroid tissue are glucose intolerant and become obese and insulin resistant with age, indicating that endogenous EPO regulates fat mass [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ]. Females exhibit an earlier onset of obesity and insulin resistance with a greater proportionate increase in fat mass. In wild-type mice, EPO-stimulated erythropoiesis is accompanied by loss of fat mass and body weight on normal chow and reduced fat mass accumulation and protection against obesity on a high-fat diet only in males (Figure 1) [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Zhang Y
      • Rogers HM
      • Zhang X
      • Noguchi CT
      Sex difference in mouse metabolic response to erythropoietin.
      ,
      • Katz O
      • Stuible M
      • Golishevski N
      • et al.
      Erythropoietin treatment leads to reduced blood glucose levels and body mass: insights from murine models.
      ]. Only male mice manifest EPO-stimulated expression of mitochondrial oxidative genes in white adipose tissue. This sex-dimorphic EPO regulation of fat mass is related to estrogen production in female mice, which regulates glucose and lipid metabolism and obesity [
      • Barros RP
      • Gustafsson JA
      Estrogen receptors and the metabolic network.
      ]. Depletion of endogenous estrogen by ovariectomy in female mice results in increased fat mass accumulation during 3 weeks of a high-fat-diet. Fat mass is reduced by EPO treatment and even more with estradiol supplementation, which was not further enhanced by the combination of EPO and estradiol (Figure 1) [
      • Zhang Y
      • Rogers HM
      • Zhang X
      • Noguchi CT
      Sex difference in mouse metabolic response to erythropoietin.
      ]. This indicates the greater protective effect of estrogen compared with EPO during diet-induced obesity and the interference of estrogen with EPO regulation of fat mass in female mice. The EPO-stimulated increase in hematocrit was comparable with and without ovariectomy, adding evidence that EPO regulation of fat mass is independent of EPO erythropoietic activity.

      EPO regulation of bone marrow adipocytes and bone

      Bone marrow adipocytes have an origin and function distinct from those of white and brown adipose tissue, increase with age and obesity, and at age 25 constitute 50% to 70% of human adult bone marrow volume and about 10% of total fat mass [
      • Suchacki KJ
      • Cawthorn WP
      • Rosen CJ
      Bone marrow adipose tissue: formation, function and regulation.
      ,
      • Schwartz AV
      Marrow fat and bone: review of clinical findings.
      ,
      • Ambrosi TH
      • Scialdone A
      • Graja A
      • et al.
      Adipocyte accumulation in the bone marrow during obesity and aging impairs stem cell-based hematopoietic and bone regeneration.
      ,
      • Fazeli PK
      • Horowitz MC
      • MacDougald OA
      • et al.
      Marrow fat and bone—new perspectives.
      ,
      • Li Y
      • Meng Y
      • Yu X
      The unique metabolic characteristics of bone marrow adipose tissue.
      ]. Bone marrow adipose tissue negatively regulates hematopoiesis, and in mice, hematopoietic recovery after chemotherapy improved with inhibition of bone marrow adipocytes by the PPAR-γ inhibitor [
      • Naveiras O
      • Nardi V
      • Wenzel PL
      • Hauschka PV
      • Fahey F
      • Daley GQ
      Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment.
      ,
      • Zhu RJ
      • Wu MQ
      • Li ZJ
      • Zhang Y
      • Liu KY
      Hematopoietic recovery following chemotherapy is improved by BADGE-induced inhibition of adipogenesis.
      ]. Bone marrow stromal cells contribute to maintenance of the hematopoietic microenvironment and regulate differentiation of bone-resorbing osteoclasts [
      • Bianco P
      • Robey PG
      Skeletal stem cells.
      ]. Bone marrow stromal cells also include nonhematopoietic progenitors for bone growth and remodeling that can differentiate into bone marrow adipocytes or bone-forming osteoblasts. Pathologies of bone loss are often associated with fatty marrow, and dysregulation of the balance between bone marrow stromal cell-derived adipogenesis and osteogenesis contributes to aging and osteoporosis [
      • Chen Q
      • Shou P
      • Zheng C
      • et al.
      Fate decision of mesenchymal stem cells: adipocytes or osteoblasts?.
      ]. Epor is expressed on a variety of cells in bone marrow: erythroid/hematopoietic cells, bone remodeling osteoclasts and osteoblasts, bone marrow adipocytes, and bone marrow stromal cells that differentiate into osteoblasts, bone marrow adipocytes, and chondrocytes. Endogenous EPO regulates bone marrow adipocytes as well as white adipose tissue, and during bone development, EPO signaling maintains the normal balance between osteogenesis and adipogenesis in the bone marrow [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ]. ΔEPORE mice with Epor restricted to erythroid tissue exhibit a two- to threefold increase in adipocyte number in bone marrow and concomitant reduction in trabecular bone, indicating a shift in bone marrow stromal cell differentiation toward adipogenesis and reduced osteogenesis [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ].
      With EPO treatment, accompanying the increase in EPO-stimulated erythropoiesis are reductions in bone marrow adipocytes and bone loss in male and female mice, independent of change in fat mass in white adipose tissue [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ,
      • Shiozawa Y
      • Jung Y
      • Ziegler AM
      • et al.
      Erythropoietin couples hematopoiesis with bone formation.
      ,
      • Singbrant S
      • Russell MR
      • Jovic T
      • et al.
      Erythropoietin couples erythropoiesis, B-lymphopoiesis, and bone homeostasis within the bone marrow microenvironment.
      ,
      • Hiram-Bab S
      • Liron T
      • Deshet-Unger N
      • et al.
      Erythropoietin directly stimulates osteoclast precursors and induces bone loss.
      ]. PPAR-γ, expressed predominantly in adipose tissue, is central to regulation of adipocyte gene expression and differentiation [
      • Brun RP
      • Tontonoz P
      • Forman BM
      Differential activation of adipogenesis by multiple PPAR isoforms.
      ]. EPO treatment reduces PPAR-γ expression in bone marrow stromal cells, which contributes to reduced bone marrow adipogenesis [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ]. Transgenic mice expressing high human EPO levels also exhibit reduced bone marrow adipocytes and trabecular and cortical bone with increased numbers of bone-resorbing osteoclasts [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ,
      • Hiram-Bab S
      • Liron T
      • Deshet-Unger N
      • et al.
      Erythropoietin directly stimulates osteoclast precursors and induces bone loss.
      ,
      • Rauner M
      • Franke K
      • Murray M
      • et al.
      Increased EPO levels are associated with bone loss in mice lacking PHD2 in EPO-producing cells.
      ]. These mice yield osteoblasts and osteoclasts that produce human EPO with increased differentiation potential, consistent with premature differentiation reducing endogenous trabecular bone, and increased alkaline phosphatase expression and mineralization [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ]. Conversely, osteoblasts from ΔEPORE mice that lack endogenous EPO signaling exhibit reduced alkaline phosphatase expression and mineralization [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ]. Osteoblasts exhibit EPO producing potential, raising the possibility for autocrine-regulated EPO response [
      • Rankin EB
      • Wu C
      • Khatri R
      • et al.
      The HIF signaling pathway in osteoblasts directly modulates erythropoiesis through the production of EPO.
      ]. EPO treatment of mesenchymal stem cell cultures increased bone mineralization in cells from young healthy human donors but not in cultures from older healthy donors, suggesting an age-dependent response [
      • Balaian E
      • Wobus M
      • Weidner H
      • et al.
      Erythropoietin inhibits osteoblast function in myelodysplastic syndromes via the canonical Wnt pathway.
      ]. EPO activity to increase osteoblast differentiation may contribute to bone loss and affect bone health by limiting osteogenic expansion. Elevated levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) have been linked to greater risk of fractures in elderly men, especially among individuals with chronic kidney disease [
      • Mirza MA
      • Karlsson MK
      • Mellstrom D
      • et al.
      Serum fibroblast growth factor-23 (FGF-23) and fracture risk in elderly men.
      ,
      • Lane NE
      • Parimi N
      • Corr M
      • et al.
      Association of serum fibroblast growth factor 23 (FGF23) and incident fractures in older men: the Osteoporotic Fractures in Men (MrOS) study.
      ]. EPO-stimulated FGF23 production in hematopoietic stem cells was associated with an increase in serum FGF23 and reduced serum phosphate, suggesting a possible mechanism of EPO-induced bone reduction caused by disrupted mineralization [
      • Clinkenbeard EL
      • Hanudel MR
      • Stayrook KR
      • et al.
      Erythropoietin stimulates murine and human fibroblast growth factor-23, revealing novel roles for bone and bone marrow.
      ].
      Although increased bone mineral density in postmenopausal obese women initially suggested obesity as a protective factor for osteoporosis, obesity was also associated with reduced bone strength and increased fracture risk [
      • Albala C
      • Yanez M
      • Devoto E
      • Sostin C
      • Zeballos L
      • Santos JL
      Obesity as a protective factor for postmenopausal osteoporosis.
      ,
      • Ishii S
      • Cauley JA
      • Greendale GA
      • et al.
      Pleiotropic effects of obesity on fracture risk: the Study of Women's Health Across the Nation.
      ,
      • Compston JE
      • Watts NB
      • Chapurlat R
      • et al.
      Obesity is not protective against fracture in postmenopausal women: GLOW.
      ]. Increased visceral and bone marrow fat in obese men was associated with impaired bone microarchitecture and mechanical properties [
      • Bredella MA
      • Lin E
      • Gerweck AV
      • et al.
      Determinants of bone microarchitecture and mechanical properties in obese men.
      ]. Obese mice with increased bone marrow adiposity exhibited increased inflammatory cytokine production, osteoclast number, and bone resorption, linking increased inflammation in response to increased marrow adiposity with osteoclastogenesis and bone resorption [
      • Halade GV
      • El Jamali A
      • Williams PJ
      • Fajardo RJ
      • Fernandes G
      Obesity-mediated inflammatory microenvironment stimulates osteoclastogenesis and bone loss in mice.
      ]. Beyond simply filling marrow space, bone marrow adipocytes negatively regulate hematopoiesis, raising the possibility that reducing marrow adipogenesis may promote hematopoietic transplant recovery [
      • Naveiras O
      • Nardi V
      • Wenzel PL
      • Hauschka PV
      • Fahey F
      • Daley GQ
      Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment.
      ]. In obese mice, short-term EPO treatment (10 days) increased hematocrit; did not affect body mass but decreased bone marrow adipocytes fivefold; reduced trabecular bone without further increase in osteoclast number; and maintained cortical bone mineral density and volume [
      • Suresh S
      • Alvarez JC
      • Dey S
      • Noguchi CT
      Erythropoietin-induced changes in bone and bone marrow in mouse models of diet-induced obesity.
      ]. While EPO administration in nonobese mice reduced bone marrow cellularity, decreased hematopoietic CD45+ cells, and increased the percentage of bone marrow erythroid cells, these parameters remained unchanged with EPO treatment in obese mice. EPO did not affect cortical bone or the increased bone marrow stromal cells in obese mice [
      • Suresh S
      • Alvarez JC
      • Dey S
      • Noguchi CT
      Erythropoietin-induced changes in bone and bone marrow in mouse models of diet-induced obesity.
      ,
      • Wu CL
      • Diekman BO
      • Jain D
      • Guilak F
      Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.
      ], perhaps in support of the need for maintenance of cortical bone to accommodate the increased body weight and resultant mechanical stress. In bone, osteoblast precursors reach bone formation sites by moving through proximal blood vessels, and the decrease in bone marrow endothelial cells in obese individuals is proposed to reduce vasculature [
      • Maes C
      • Kobayashi T
      • Selig MK
      • et al.
      Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels.
      ,
      • McGuire TR
      • Brusnahan SK
      • Bilek LD
      • et al.
      Inflammation associated with obesity: relationship with blood and bone marrow endothelial cells.
      ]. The reduction in bone marrow endothelial cells in obese mice is reversed with EPO treatment [
      • Suresh S
      • Alvarez JC
      • Dey S
      • Noguchi CT
      Erythropoietin-induced changes in bone and bone marrow in mouse models of diet-induced obesity.
      ], and may contribute to increased vasculature and bone repair.
      EPO-stimulated bone remodeling is context dependent. In rodent models of bone fracture repair, EPO stimulated early endochondral ossification and bone mineralization, accelerated bone healing, inhibited bone resorption and reduced osteoclasts, increased endosteal vascularization, and reduced NFκB expression [
      • Holstein JH
      • Menger MD
      • Scheuer C
      • et al.
      Erythropoietin (EPO): EPO-receptor signaling improves early endochondral ossification and mechanical strength in fracture healing.
      ,
      • Garcia P
      • Speidel V
      • Scheuer C
      • et al.
      Low dose erythropoietin stimulates bone healing in mice.
      ,
      • Li C
      • Shi C
      • Kim J
      • et al.
      Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling.
      ,
      • Mihmanli A
      • Dolanmaz D
      • Avunduk MC
      • Erdemli E
      Effects of recombinant human erythropoietin on mandibular distraction osteogenesis.
      ,
      • Omlor GW
      • Kleinschmidt K
      • Gantz S
      • Speicher A
      • Guehring T
      • Richter W
      Increased bone formation in a rabbit long-bone defect model after single local and single systemic application of erythropoietin.
      ]. Animal models of bone injury suggest the potential for EPO to recruit bone marrow stromal cells with bone-repairing ability to enhance bone regeneration or accelerate bone morphogenic protein 2 healing activity [
      • Li J
      • Huang Z
      • Li B
      • Zhang Z
      • Liu L
      Mobilization of transplanted bone marrow mesenchymal stem cells by erythropoietin facilitates the reconstruction of segmental bone defect.
      ,
      • Nair AM
      • Tsai YT
      • Shah KM
      • et al.
      The effect of erythropoietin on autologous stem cell-mediated bone regeneration.
      ,
      • Sun H
      • Jung Y
      • Shiozawa Y
      • Taichman RS
      • Krebsbach PH
      Erythropoietin modulates the structure of bone morphogenetic protein 2-engineered cranial bone.
      ]. In a pilot study of patients with tibiofibular fractures, it was suggested that EPO injection at the fracture site 2 weeks after surgery promotes faster union by 2 weeks and a lower rate of nonunion fracture [
      • Bakhshi H
      • Kazemian G
      • Emami M
      • Nemati A
      • Karimi Yarandi H
      • Safdari F
      Local erythropoietin injection in tibiofibular fracture healing.
      ].

      EPO regulates bone marrow stromal cell differentiation

      Mouse models of ectopic ossification illustrated the potential for EPO to regulate bone marrow stromal cell differentiation to osteoblastic or adipogenic lineages and to recapitulate endogenous formation of bone and bone marrow adipocytes [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ]. Transplantation of collagen sponges containing bone marrow stromal cells into immunodeficient mice resulted in ossicle formation consisting of bone, adipocytes, and stroma of donor origin and hematopoiesis from the recipient [
      • Robey PG
      • Kuznetsov SA
      • Riminucci M
      • Bianco P
      Bone marrow stromal cell assays: in vitro and in vivo.
      ]. The bone ossicles mimicked the changes in endogenous bone and bone marrow adipocyte formation of donor mice with altered EPO signaling. For bone marrow stromal cells from transgenic mice expressing high EPO, ossicle formation was significantly attenuated with a marked decrease in marrow adipocytes and greater than 10-fold reduction in bone and a lack of well-defined trabecular and cortical bone [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ]. Bone marrow stromal cells from ΔEPORE mice that lack EPO signaling produced ossicles with reduced bone formation and a greater than twofold increase in marrow adipocytes.
      In mice with targeted deletion of Epor in osteoblasts, trabecular bone is reduced by more than 20% by 12 weeks of age without change in the numbers of osteoblasts, osteoclasts, and marrow adipocytes, and osteogenic cultures exhibited reduced differentiation and mineralization [
      • Suresh S
      • Lee J
      • Noguchi CT
      Erythropoietin signaling in osteoblasts is required for normal bone formation and for bone loss during erythropoietin-stimulated erythropoiesis.
      ]. Like ΔEPORE mice, mice with osteoblast deletion of Epor manifested no additional bone loss with EPO treatment, indicating that bone loss requires a direct osteoblast EPO response and is not related to EPO-stimulated erythropoiesis [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ,
      • Suresh S
      • Lee J
      • Noguchi CT
      Erythropoietin signaling in osteoblasts is required for normal bone formation and for bone loss during erythropoietin-stimulated erythropoiesis.
      ]. Receptor activator of nuclear factor κΒ ligand (RANKL) made in osteoblasts, bone marrow stromal cells, and B and T lymphocytes contributes to bone remodeling by activating osteoclasts via binding to its receptor (RANK) to promote bone resorption [
      • Boyce BF
      • Xing L
      Functions of RANKL/RANK/OPG in bone modeling and remodeling.
      ]. In bone marrow B cells, EPO increased RANKL expression, and knockdown of Epor increased trabecular and cortical bone mass and decreased trabecular bone loss with EPO treatment [
      • Deshet-Unger N
      • Kolomansky A
      • Ben-Califa N
      • et al.
      Erythropoietin receptor in B cells plays a role in bone remodeling in mice.
      ].

      EPO reduces inflammation in white adipose tissue in obese mice

      EPO protection against inflammation reduces pro-inflammatory cytokine response and macrophage infiltration and has been observed in animal models of tissue injury, including adult and preterm brain, acute and chronic heart injury, and chemical-induced colitis mediated in part by JAK2, STAT, and AKT activation [
      • Villa P
      • Bigini P
      • Mennini T
      • et al.
      Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis.
      ,
      • Wassink G
      • Davidson JO
      • Dhillon SK
      • et al.
      Partial white and gray matter protection with prolonged infusion of recombinant human erythropoietin after asphyxia in preterm fetal sheep.
      ,
      • Rui T
      • Feng Q
      • Lei M
      • et al.
      Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1.
      ,
      • Li Y
      • Takemura G
      • Okada H
      • et al.
      Reduction of inflammatory cytokine expression and oxidative damage by erythropoietin in chronic heart failure.
      ,
      • Nairz M
      • Schroll A
      • Moschen AR
      • et al.
      Erythropoietin contrastingly affects bacterial infection and experimental colitis by inhibiting nuclear factor-kappaB-inducible immune pathways.
      ]. In mouse models, EPO decreased hypoxic and inflammatory response in sepsis-induced acute kidney injury and suppressed macrophage foam cell formation in cardiovascular disease [
      • Stoyanoff TR
      • Rodriguez JP
      • Todaro JS
      • Colavita JPM
      • Torres AM
      • Aguirre MV
      Erythropoietin attenuates LPS-induced microvascular damage in a murine model of septic acute kidney injury.
      ,
      • Lu KY
      • Ching LC
      • Su KH
      • et al.
      Erythropoietin suppresses the formation of macrophage foam cells: role of liver X receptor alpha.
      ]. In white adipose tissue, macrophages in the stromal vascular fraction contribute to metabolic homeostasis [
      • Bowles AC
      • Wise RM
      • Gerstein BY
      • et al.
      Immunomodulatory effects of adipose stromal vascular fraction cells promote alternative activation macrophages to repair tissue damage.
      ]. White adipose tissue in obese mice shifts toward a pro-inflammatory state with increased macrophage infiltration, M2-like pro-inflammatory subtype, and inflammatory cytokine production [
      • Cinti S
      • Mitchell G
      • Barbatelli G
      • et al.
      Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.
      ]. This is characterized by the appearance of crownlike structures, which are histological features of inflammatory adipose tissues of obese animals consisting of macrophages surrounding necrotic adipocytes [
      • Cinti S
      • Mitchell G
      • Barbatelli G
      • et al.
      Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.
      ].
      In obese mice, 2 weeks of EPO treatment increases hematocrit without change in fat mass, but improves glucose tolerance and insulin sensitivity and shifts obesity-associated white adipose tissue inflammation toward an anti-inflammatory state [
      • Alnaeeli M
      • Raaka BM
      • Gavrilova O
      • Teng R
      • Chanturiya T
      • Noguchi CT
      Erythropoietin signaling: a novel regulator of white adipose tissue inflammation during diet-induced obesity.
      ,
      • Alnaeeli M
      • Noguchi CT
      Erythropoietin and obesity-induced white adipose tissue inflammation: redefining the boundaries of the immunometabolism territory.
      ]. EPO administration reduces white adipose tissue macrophage infiltration, crownlike structures, expression of pro-inflammatory cytokines, and production of tumor necrosis factor (TNF)-α, and increases anti-inflammatory cytokine interleukin (IL)-10 production. Macrophages respond directly to EPO stimulation with increased STAT3 activation and reduced inducible nitric oxide synthase (iNOS) and IL-1β expression. EPO treatment shifts the macrophage population toward an anti-inflammatory subtype that requires IL-4 and STAT6 activity, indicating that EPO contributes to local macrophage subtype polarization [
      • Alnaeeli M
      • Raaka BM
      • Gavrilova O
      • Teng R
      • Chanturiya T
      • Noguchi CT
      Erythropoietin signaling: a novel regulator of white adipose tissue inflammation during diet-induced obesity.
      ]. Endogenous EPO also provides immune modulatory activity. On a high-fat diet, weight gain and obesity are comparable in ΔEPORE mice with Epor restricted to erythroid tissue and control mice, but ΔEPORE mice exhibit a greater inflammatory response in adipose tissue [
      • Alnaeeli M
      • Raaka BM
      • Gavrilova O
      • Teng R
      • Chanturiya T
      • Noguchi CT
      Erythropoietin signaling: a novel regulator of white adipose tissue inflammation during diet-induced obesity.
      ]. ΔEPORE white adipose tissue exhibits denser macrophage infiltration and increased crownlike structures, inflammatory chemokine expression in the stromal vascular fraction, TNF-α production, and circulating inflammatory monocytes. These mice have greater glucose intolerance and insulin resistance that are unchanged with EPO treatment [
      • Alnaeeli M
      • Raaka BM
      • Gavrilova O
      • Teng R
      • Chanturiya T
      • Noguchi CT
      Erythropoietin signaling: a novel regulator of white adipose tissue inflammation during diet-induced obesity.
      ].
      In addition to adipocyte response to EPO [
      • Pan Y
      • Shu JL
      • Gu HF
      • et al.
      Erythropoietin improves insulin resistance via the regulation of its receptor-mediated signaling pathways in 3T3L1 adipocytes.
      ], macrophage inflammatory response in white adipose tissue during obesity influences insulin resistance [
      • Lumeng CN
      • Bodzin JL
      • Saltiel AR
      Obesity induces a phenotypic switch in adipose tissue macrophage polarization.
      ,
      • Liu R
      • Nikolajczyk BS
      Tissue immune cells fuel obesity-associated inflammation in adipose tissue and beyond.
      ], further linking erythropoietin metabolic response with improved insulin sensitivity. Other organs contributing to EPO activity during diet-induced obesity include a JAK2-dependent EPO-protective effect on insulin-producing pancreatic β cells, inducing pancreatic islets and proliferative anti-inflammatory and angiogenic activity in diabetic mouse models [
      • Choi D
      • Schroer SA
      • Lu SY
      • et al.
      Erythropoietin protects against diabetes through direct effects on pancreatic beta cells.
      ]. In liver, EPO enhances AKT activation and reduces obesity-associated gluconeogenesis and liver inflammation in obese mice [
      • Meng R
      • Zhu D
      • Bi Y
      • Yang D
      • Wang Y
      Erythropoietin inhibits gluconeogenesis and inflammation in the liver and improves glucose intolerance in high-fat diet-fed mice.
      ]. EPO also exerts a neuroendocrine response in mice affecting metabolic homeostasis [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Dey S
      • Li X
      • Teng R
      • et al.
      Erythropoietin regulates POMC expression via STAT3 and potentiates leptin response.
      ].

      EPO regulates hypothalamus production of pro-opiomelanocortin

      EPO treatment in male mice increases locomotor activity and decreases food intake to promote a lean phenotype, decreasing body weight and fat mass [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ]. Regulation of appetite by the hypothalamus is mediated by neurons in the arcuate nucleus that sense changes in nutrient status. Stimulation of neurons that produce neuropeptide Y and agouti-related protein increase appetite, while activation of neurons that produce pro-opiomelanocortin (POMC) suppresses appetite. Hypothalamus Epor expression localizes to POMC neurons, and EPO administration increases POMC in the hypothalamus and in primary hypothalamus neural cell cultures, but not expression of neuropeptide Y or agouti-related protein [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Dey S
      • Li X
      • Teng R
      • et al.
      Erythropoietin regulates POMC expression via STAT3 and potentiates leptin response.
      ]. EPO stimulates STAT3 activation in the hypothalamus and POMC neuron cultures, and ΔEPORE mice exhibit decreased hypothalamus STAT3 activation and POMC production [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Dey S
      • Li X
      • Teng R
      • et al.
      Erythropoietin regulates POMC expression via STAT3 and potentiates leptin response.
      ].
      The hypothalamic–pituitary axis contributes importantly to the balance between energy intake and energy expenditure to maintain metabolic homeostasis through secretion of endocrine hormones [
      • Lemche E
      • Chaban OS
      • Lemche AV
      Neuroendorine and epigentic mechanisms subserving autonomic imbalance and HPA dysfunction in the metabolic syndrome.
      ,
      • Dey S
      • Noguchi CT
      Erythropoietin and hypothalamic–pituitary axis.
      ]. In the hypothalamus, EPO increases POMC production, while in the pituitary, EPO decreases cytosolic calcium-dependent POMC-derived adrenocorticotropic hormone (ACTH) secretion [
      • Dey S
      • Scullen T
      • Noguchi CT
      Erythropoietin negatively regulates pituitary ACTH secretion.
      ,
      • Tse A
      • Lee AK
      • Tse FW
      Ca2+ signaling and exocytosis in pituitary corticotropes.
      ]. In contrast, ΔEPORE mice that lack EPO signaling in nonhematopoietic tissue are obese and exhibit reduced hypothalamus POMC production and an elevated plasma concentration of ACTH [
      • Teng R
      • Gavrilova O
      • Suzuki N
      • et al.
      Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.
      ,
      • Dey S
      • Scullen T
      • Noguchi CT
      Erythropoietin negatively regulates pituitary ACTH secretion.
      ]. The metabolic changes observed in ΔEPORE mice provide evidence that the activity of endogenous EPO in the hypothalamic–pituitary axis contributes to neuroendocrine regulation of metabolism and obesity [
      • Dey S
      • Noguchi CT
      Erythropoietin and hypothalamic–pituitary axis.
      ].

      Cerebral EPO protects against diet-induced obesity

      Transgenic mice overexpressing brain-specific human EPO without affecting hematocrit (Tg21 mice) [
      • Wiessner C
      • Allegrini PR
      • Ekatodramis D
      • Jewell UR
      • Stallmach T
      • Gassmann M
      Increased cerebral infarct volumes in polyglobulic mice overexpressing erythropoietin.
      ] have improved glucose tolerance on normal chow and high-fat diet and increased insulin sensitivity during a high-fat diet [
      • Dey S
      • Cui Z
      • Gavrilova O
      • Zhang X
      • Gassmann M
      • Noguchi CT
      Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.
      ]. Cerebral EPO exhibits a gender-specific response in high-fat-diet obesity, and male but not female Tg21 mice exhibit resistance to obesity, reduced fat mass accumulation, and higher energy expenditure [
      • Dey S
      • Cui Z
      • Gavrilova O
      • Zhang X
      • Gassmann M
      • Noguchi CT
      Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.
      ]. Overnutrition promotes hypothalamus inflammation with activation of microglial cells, specialized macrophage cells in the brain, and increased pro-inflammatory cytokines before overt obesity and inflammation in white adipose tissue [
      • Valdearcos M
      • Xu AW
      • Koliwad SK
      Hypothalamic inflammation in the control of metabolic function.
      ]. Transmembrane TNF-α is expressed on activated macrophages, lymphocytes, and other cell types (TNF-α+ cells) and undergoes proteolytic cleavage to release the soluble form of TNF-α [
      • Horiuchi T
      • Mitoma H
      • Harashima S
      • Tsukamot H
      • Shimoda T
      Transmembrane TNF-alpha: structure, function and interaction with anti-TNF agents.
      ]. Male Tg21 mice on a high-fat diet exhibit reduced levels of hypothalamus-activated microglial cells, TNF-α+ cells, inflammatory cytokine gene expression, recruitment of blood myeloid monocyte-derived cells, and reduced serum ACTH and corticosterone (Figure 2) [
      • Dey S
      • Cui Z
      • Gavrilova O
      • Zhang X
      • Gassmann M
      • Noguchi CT
      Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.
      ]. Increased cerebral EPO via an intracerebroventricular pump in male wild-type mice on a high-fat diet also exhibited decrease weight gain and reduced fat mass accumulation and, in the hypothalamus, reduced inflammatory cytokine expression and increased anti-inflammatory IL-10 expression (Figure 2) [
      • Dey S
      • Cui Z
      • Gavrilova O
      • Zhang X
      • Gassmann M
      • Noguchi CT
      Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.
      ]. In contrast, male mice with targeted deletion of Epor in neural cells gained more weight on a high-fat diet, were more glucose intolerant, and exhibited greater induction of hypothalamus TNF-α, activated microglial cells, and recruitment of peripheral myeloid cells [
      • Dey S
      • Cui Z
      • Gavrilova O
      • Zhang X
      • Gassmann M
      • Noguchi CT
      Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.
      ].
      Figure 2
      Figure 2Cerebral EPO protects against high fat diet-induced hypothalamus inflammation. C57BL/6 male mice fed a high-fat diet become obese, and the obesity is accompanied by hypothalamic inflammation and associated microglial cell activation. With implantation of an EPO-secreting intracerebroventricular pump or by generation of transgenic Tg21 mice that express human EPO in brain, elevated cerebral EPO decreased susceptibility to diet-induced obesity and protected against obesity-associated hypothalamic inflammation. Mice with an implanted EPO intracerebroventricular pump and Tg21 mice exhibit a normal hematocrit because of limited transport of secreted or transgenic EPO across the blood–brain barrier. Estrogen provides protection against diet-induced obesity and associated hypothalamic inflammation in female wild-type and Tg21 mice. Ovariectomy in female mice abrogates the estrogen anti-obesity activity, and ovariectomized mice fed a high-fat diet become obese with concomitant hypothalamic inflammation. Ovariectomized Tg21 mice with elevated cerebral EPO exhibit reduced susceptibility to diet-induced obesity and protection against hypothalamic inflammation.
      The sex-dimorphic response of Tg21 mice to high fat diet-induced obesity provides another illustration of estrogen protective activity against diet-induced obesity in female mice that suppresses EPO metabolic activity in fat mass regulation as well as associated hypothalamus inflammation. With ovariectomy, which blocks anti-obesity estrogen activity, female Tg21 mice exhibited the protective effect of cerebral EPO, and only wild-type female mice exhibited increased fat mass and hypothalamus inflammation, microglial activation, and inflammatory cytokine expression (Figure 2) [
      • Dey S
      • Cui Z
      • Gavrilova O
      • Zhang X
      • Gassmann M
      • Noguchi CT
      Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation.
      ].

      Conclusions

      Animal models illustrate that both endogenous EPO and exogenous EPO contribute to metabolic response. Epor expression in white adipose tissue, adipocytes, and macrophages, and in brain, neurons, and microglia, mediates EPO regulation of glucose metabolism, insulin sensitivity, fat mass, and obesity-related inflammation. A demonstrated gender-specific ventilatory response in mice with hypoxia induction of EPO is sensitive to ovarian steroids [
      • Soliz J
      • Khemiri H
      • Caravagna C
      • Seaborn T
      Erythropoietin and the sex-dimorphic chemoreflex pathway.
      ]. Similarly, estrogen anti-obesity activity in female mice contributes to the EPO sex-dimorphic metabolic response and EPO activity in adipose tissue and brain to regulate fat mass, and obesity-related inflammation is observed only in male mice. Secondary analysis of full-heritage Pima Indians from the Gila River Indian Community with a high prevalence of obesity and type 2 diabetes [
      • Smith CJ
      • Nelson RG
      • Hardy SA
      • Manahan EM
      • Bennett PH
      • Knowler WC
      Survey of the diet of Pima Indians using quantitative food frequency assessment and 24-hour recall. Diabetic Renal Disease Study.
      ,
      • Pavkov ME
      • Hanson RL
      • Knowler WC
      • Bennett PH
      • Krakoff J
      • Nelson RG
      Changing patterns of type 2 diabetes incidence among Pima Indians.
      ] reveals endogenous EPO levels associated negatively with hemoglobin and, in males, negatively with percentage weight change per year compared with the positive association observed in females [
      • Reinhardt M
      • Dey S
      • Tom Noguchi C
      • Zhang Y
      • Krakoff J
      • Thearle MS
      Non-hematopoietic effects of endogenous erythropoietin on lean mass and body weight regulation.
      ]. These gender-specific relationships between EPO level and body weight are consistent with reduction of body weight with EPO treatment only in male mice and ovariectomized female mice [
      • Zhang Y
      • Rogers HM
      • Zhang X
      • Noguchi CT
      Sex difference in mouse metabolic response to erythropoietin.
      ].
      EPO regulation of bone marrow adipocytes and skeletal bone formation is not gender specific and is mediated by Epor in bone marrow stromal cells, osteoblasts, adipocytes, and osteoclasts [
      • Suresh S
      • de Castro LF
      • Dey S
      • Robey PG
      • Noguchi CT
      Erythropoietin modulates bone marrow stromal cell differentiation.
      ,
      • Suresh S
      • Lee J
      • Noguchi CT
      Erythropoietin signaling in osteoblasts is required for normal bone formation and for bone loss during erythropoietin-stimulated erythropoiesis.
      ]. In mice, endogenous EPO is required for normal bone development and regulation of bone marrow adipocytes, while continuous EPO treatment to stimulate erythropoiesis decreases bone formation and marrow adiposity, providing implications for bone health in erythropoietic pathologies with elevated EPO such as thalassemia, sickle cell disease, and polycythemia vera [
      • Vichinsky EP
      The morbidity of bone disease in thalassemia.
      ,
      • Sarrai M
      • Duroseau H
      • D'Augustine J
      • Moktan S
      • Bellevue R
      Bone mass density in adults with sickle cell disease.
      ,
      • Farmer S
      • Horvath-Puho E
      • Vestergaard H
      • Hermann AP
      • Frederiksen H
      Chronic myeloproliferative neoplasms and risk of osteoporotic fractures: a nationwide population-based cohort study.
      ]. Assessment of elderly men with normal kidney function in Sweden revealed that a high EPO level was associated with higher fracture risk independent of hemoglobin and age [
      • Kristjansdottir HL
      • Lewerin C
      • Lerner UH
      • et al.
      High plasma erythropoietin predicts incident fractures in elderly men with normal renal function: The MrOS Sweden Cohort.
      ]. New pharmacological approaches to stimulate EPO activity, such as the prolyl hydroxylase inhibitors [
      • Chen N
      • Hao C
      • Liu BC
      • et al.
      Roxadustat treatment for anemia in patients undergoing long-term dialysis.
      ,
      • Chen N
      • Hao C
      • Peng X
      • et al.
      Roxadustat for anemia in patients with kidney disease not receiving dialysis.
      ], may provide the methodology to selectively increase erythropoiesis while maintaining bone health or to promote a tissue-specific nonhematopoietic response without increased erythropoiesis.

      Acknowledgments

      This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases.

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