Aromatase deficiency in hematopoietic cells improves glucose tolerance in male mice through skeletal muscle-specific effects.

Estrogens are important for maintaining metabolic health in males. However, the key sources of local estrogen production for regulating energy metabolism have not been fully defined. Immune cells exhibit aromatase activity and are resident in metabolic tissues. To determine the relative contribution of immune cell-derived estrogens for metabolic health in males, C57BL6/J mice underwent bone marrow transplant with marrow from either wild-type (WT(WT)) or aromatase-deficient (WT(ArKO)) donors. Body weight, body composition, and glucose and insulin tolerance were assessed over 24 weeks with mice maintained on a regular chow diet. No differences were found in insulin sensitivity between groups, but WT(ArKO) mice were more glucose tolerant than WT(WT) mice 20 weeks after transplant, suggestive of enhanced glucose disposal (AUCglucose 6061±3349 in WT(WT) mice versus 3406±1367 in WT(ArKO) mice, p = 0.01). Consistent with this, skeletal muscle from WT(ArKO) mice showed higher expression of the mitochondrial genes Ppargc1a (p = 0.03) and Nrf1 (p = 0.01), as well as glucose transporter type 4 (GLUT4, Scl2a4; p = 0.02). Skeletal muscle from WT(ArKO) mice had a lower concentration of 17β-estradiol (5489±2189 pg/gm in WT(WT) mice versus 3836±2160 pg/gm in WT(ArKO) mice, p = 0.08) but higher expression of estrogen receptor-α (ERα, Esr1), raising the possibility that aromatase deficiency in immune cells led to a compensatory increase in ERα signaling. No differences between groups were found with regard to body weight, adiposity, or gene expression within adipose tissue or liver. Immune cells are a key source of local 17β-estradiol production and contribute to metabolic regulation in males, particularly within skeletal muscle. The respective intracrine and paracrine roles of immune cell-derived estrogens require further delineation, as do the pathways that regulate aromatase activity in immune cells specifically within metabolic tissues.

Introduction

Estrogens are now recognized to play important metabolic roles in men. Initial evidence that men require adequate estrogen exposure for metabolic health derived from men with rare genetic syndromes of estrogen deficiency. Thus, men with loss of either functional estrogen receptor-α (ERα) or aromatase—the enzyme that generates estrogens from androgen precursors–exhibit metabolic derangements including increased visceral adiposity, insulin resistance, and reduced bone mineral density [1, 2]. More recently, clinical intervention studies have shown that short-term estrogen deprivation in men results in increased adiposity [3, 4] and reduced insulin sensitivity [5]. In parallel with these clinical observations, genetic mouse models also have demonstrated the importance of estrogens for regulating energy balance, body composition, insulin sensitivity, and skeletal health in males [6-8]. Similar to men with estrogen insufficiency, male mice with global aromatase deficiency exhibit increased adiposity, insulin resistance, and lower bone mass [9, 10]. However, the mechanisms underlying estrogen-mediated metabolic regulation in males remain incompletely understood, and the key tissue-specific sites of estrogen action, as well as local sources of estrogen production, have to yet be clearly defined.

Estrogens are generated not only from classically steroidogenic tissues including gonads and adrenal glands but also are generated locally within tissues, and numerous cell types are able to convert androgens to estrogens through aromatase activity [11, 12]. Thus, tissue levels of estrogens, including the most important bioactive estrogen, 17β-estradiol, may be dissociated from circulating levels [13, 14]. Adipose tissue stromal cells express aromatase and are a well-established source of both local and circulating estrogens [11]. Aromatase expression and activity similarly have been demonstrated in hepatocytes, myocytes, and osteoblasts, underscoring the importance of local estrogen production in key metabolic tissues. Notably, immune cells are present in all key metabolic tissues, and aromatase activity has been identified in immune cells including macrophages and both T and B lymphocytes [15-17]. Therefore, the possibility exists that aromatase activity within immune cells generates bioactive estrogens that mediate cell-intrinsic, intracrine effects, with consequent changes in cellular phenotype and function. Further, immune cells may secrete estrogens and could thereby contribute to tissue-specific estrogen concentrations with signaling effects conferred in neighboring cells.

No studies to date have examined the metabolic effects of estrogens generated specifically within immune cells. We used a bone marrow transplant model to reconstitute hematopoietic cells in wild-type male mice with either wild-type (WT) or aromatase deficient (ArKO) hematopoietic cells. Our goal was to determine the metabolic effects of aromatase deficiency in hematopoietic cells in male mice. We predicted that loss of aromatase function selectively within hematopoietic cells would lead to increased adiposity and impaired glucose tolerance, reproducing the metabolic phenotype of male mice with global aromatase deficiency.

Materials and methods

Measurement of immune cell estrogen secretion

To isolate peritoneal immune cells, thioglycolate (BD; Franklin Lakes, NJ) was administered to male C57Bl6/J mice by intra-peritoneal injection as described previously [18]. Five days subsequent to injection, peritoneal fluid was collected by lavage. Cells were separated by centrifugation of lavage fluid and plated at a density of 2x106 per well in 6-well tissue culture plates. Macrophages were enriched through adhesion purification and cultured 24 hours in RPMI media with 10% fetal bovine serum and 1% penicillin/streptomycin.

Immune cell conditioned media was collected, and immune cell estrogen secretion was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as others have described previously [19]. In duplicate, 0.1 mL of media from each sample was transferred to glass tubes containing 25 μL of internal standard (5d-estradiol, 4d -estrone, 4pg/ul) and vortexed. Samples were held at room temperature for 10 minutes, followed by addition of 2.5 mL of hexane: ethyl Acetate (8:2 v/v). Samples were rotated for 15 min, then spun at 4500 rpm in the microcentrifuge for 15 min at 4°C. The upper solvent phase (2 mL) was transferred to a clean glass tube, and this procedure was repeated using 1.5 mL of hexane: ethyl Acetate (8:2 v/v). Hexane: ethyl acetate was then removed using a Speed-Vac with solvent trap while heating to 38°C for 30 minutes. Next, 0.3ml of hexane: ethyl Acetate (8:2 v/v) was added to sample tube and vortexed, then removed again by Speed-Vac. Sodium bicarbonate (25 μL of 100 mM, pH 10.5) and 25 μl of dansyl chloride in acetone (1 g/L) were added to sample vial, and samples were spun briefly at 1000 rpm. Finally, samples were heated to 60°C for 3 min and again briefly spun at 1000 rpm. Samples were transferred to a clean glass conical HPLC vial. The vial was then placed in 1.8ml conical MF tube and spun at 13,000 rpm for 5 minutes in microcentrifuge. Without disrupting pellet, 40 μl of sample was transferred to a clean conical HPLC vial. Samples were stored overnight at room temperature and then at 4°C. Extraction and derivatization of the standards and samples were performed simultaneously. Samples were run on an AB Sciex 5500 QTRAP tandem quadrupole mass spectrometer. Chromatographic separation performed using a Phenomenex Kinetex 1.7u 100 x 2.1 mm Phenyl-Hexyl column. The inter-assay CVs for estrone and estradiol were 12.1% and 5.6%, respectively, with a lower limit of detection of 3.6 pmol/L for both.

Animals and study design

Generation of donor mice: Male and female mice heterozygous for aromatase deficiency were generated as previously described [8, 9] and bred to generate littermate wild-type (WT) and aromatase-deficient (ArKO) male mice. The breeder mice were from a congenic C57BL6/J strain and provided from an animal colony at the University of Texas-Southwestern maintained by the senior author (OKO); these included 1 male and 2 female mice heterozygous for aromatase deficiency and 1 wild-type male mouse. The offspring served as bone marrow donors (n = 3 ArKO donor male mice and n = 4 WT donor male mice). For animal genotyping, tissue harvested through tail clip was boiled in NaOH (50 Mm, 600 μL) for 10–20 minutes then vortexed for 5–10 sec and neutralized with 1M Tris (100 μL, pH 8.0). Samples were centrifuged for 6 min at 12,000 rpm to pellet debris, and 200 μL was transferred to a fresh tube. For each sample, 1–2 μL was used for PCR in a 25 μL reaction. PCR was performed as detailed elsewhere [20] using Taq DNA polymerase (Thermo Fisher Scientific; Waltham, MA) and Redi Load gel loading buffer (Invitrogen; Carlsbad, CA). The following primer pairs were used: Neo F: 5’ ATC AGG ATG ATC TGG ACG AAG 3,’ Neo R: 5’ CCA CAG TCG ATG AAT CCA GAA 3,’ exon 9 F: 5’ GTG ACA GAG ACA TAA AGA TCG 3,’ and exon 9 R: 5’ GTA AAT TCA TTG GGC TTA GGG 3.’ PCR products were run on a 2.5% agarose gel, with product sizes of 170 bp for Neo and 220 bp for exon 9.

Bone marrow transplant procedure: Wild-type C57BL6/J male mice (n = 30) were purchased through the Jackson Laboratory (Bar Harbor, ME; strain #000664) and served as bone marrow transplant recipients. Mice were allowed to acclimate after transfer for 4 weeks and underwent transplant at 9 weeks of age (Fig 1A). Recipient mice were subject to lethal irradiation (10 Gy) and administered neomycin (2 mg/mL) in drinking water for 2 weeks post-irradiation. Recipients of bone marrow transplant were monitored 3–4 times each week for 4 weeks subsequent to irradiation until marrow engraftment occurred. Mice were monitored for any signs or behaviors indicative of poor health, including hunching, difficulty breathing, or listlessness, and any concerning signs or behaviors were reported to veterinary medicine.

Fig 1

Schematic of the study design and measurement of immune cell secretion of estrogens.

A metabolic phenotyping study was performed in wild-type male mice transplanted with bone marrow from wild-type or aromatase-deficient donors (A). Concentrations of 17β-estradiol and estrone were markedly higher in media conditioned by murine peritoneal immune cells than cell-free media (n = 8 samples conditioned media, n = 4 samples cell-free media; ***p<0.0001) (B).

The morning following irradiation, donor mice were sacrificed by CO2 inhalation and exsanguination, and femurs and tibias were harvested. Femurs and tibias were washed in ethanol once followed by 3 washes in phosphate-buffered saline (PBS). A single end of the bone was clipped, and bones were placed in 600 μL tubes punctured with an 18” needle and placed within a 1.5 mL tube. The bones were then centrifuged at 10g for 8 seconds, and bone marrow was collected in the 1.5 mL tube. Bone marrow from each tube was immediately suspended in 1 mL red blood cell lysis buffer (Sigma-Aldrich; St. Louis, MO), and marrow from donor mice of each genotype was then pooled in 50 mL tubes. To quench the lysis reaction, 3–5 mL PBS was added to each tube, and samples were centrifuged at 400g for 5 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 10 mL PBS. Cells were counted using a hemocytometer, and cells were resuspended in 1% PBS to a final concentration of 23.8x106 cells/mL. Pooled cells were aliquoted into syringes for bone marrow transplant. A total of 7x106 cells (300 μL injection volume) was administered to irradiated recipient mice through retro-orbital injection on the day following irradiation. Thus, all bone marrow transplant recipients were WT mice with marrow from either WT [WT(WT)] or ArKO [WT(ArKO)] donors.

During the post-transplant period, 3 mice [2 WT(WT), 1 WT(ArKO)] died, presumably due to infection prior to engraftment of transplanted cells. Another mouse [(WT(ArKO)] died unexpectedly during an insulin tolerance test, presumably due to hypoglycemia. Consequently, a total of 26 mice (n = 13 per group) remained and were included in the phenotyping study. During fat-water imaging for detailed body fat quantification, 2 animals in the WT(WT) group died during recovery from anesthesia. Therefore, for all post-mortem tissue analyses, a total of 24 mice were included (n = 11 WT(WT) animals and n = 13 WT(ArKO) animals). Bone marrow engraftment was verified through genotyping of circulating immune cells. At 8 weeks after transplant (study week 4), whole blood was collected and centrifuged for plasma preparation, and the supernatant was removed. NaOH (600 μL) was added to the remaining cell pellet, and samples were then placed on a heat block for 20 minutes. Genotyping was performed on 1 μL of the end product.

Body weight and food intake were measured weekly. Glucose and insulin tolerance tests were performed at study weeks 8 and 16 (12 and 20 weeks after bone marrow transplant, respectively). Body composition was measured by QMR spectroscopy at study weeks 8 and 16. Measurement of visceral, subcutaneous, and liver fat volume subsequently was performed using fat-water imaging (study week 22), and animals were sacrificed at study week 24 through cervical dislocation and exsanguination.

For the duration of the study, mice were maintained on a regular chow diet (D12450H, Research Diets, Inc; New Brunswick, NJ, USA). All mice were group housed (4–5 animals per cage, breeding pairs 2 animals per cage) with ad libitum access to food and water. In order to minimize suffering and risk to animals, fasting times were limited to 4–5 hours prior to metabolic testing, bone marrow transplant and sacrifice were performed under isoflurane anesthesia, and mice were placed on heating pads immediately following fat-water imaging. Adequacy of anesthesia was assessed through monitoring of respiratory rate and toe pinch response. All study procedures were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC, protocol #4369–01).

Immunohistochemistry and adipocyte size measurement

Antigen presenting cells in formalin-fixed, paraffin-embedded epididymal and inguinal adipose tissue were quantified using a rat monoclonal antibody against MAC2 (1:2500, Cedarlane Laboratories), as described previously [21]. Adipocyte number and cross-sectional area were measured using computer image analysis with Image J software as previously described [22].

Intra-peritoneal glucose and insulin tolerance tests

At study weeks 8 and 16, glucose homeostasis was assessed through glucose (GTT) and insulin (ITT) tolerance tests. Animals were fasted for 5 hours prior to testing and administered insulin (0.75 U/kg body weight) or glucose (1.5 g/kg body weight, 20% dextrose solution) by intra-peritoneal injection. Blood glucose was measured at baseline and 15, 30, 60, 90, and 120 minutes after injection using a hand-held glucose meter (Accu-Chek; Roche Diabetes Care, Inc; Basel, SUI).

Body composition assessment

Body composition was assessed at study weeks 8 and 16 by QMR spectroscopy performed through the University of Washington Nutrition Obesity Research Center (UW NORC) Energy Balance Core [22, 23]. At study week 22, body composition also was measured by fat-water imaging using a 3-Tesla magnetic resonance system to assess body fat distribution. This method was used to quantify total, visceral and subcutaneous fat volumes as well as liver fat content and has been previously described in detail [22]. All fat-water imaging analyses were performed by a single radiologist blinded to animal grouping.

Metabolic tissue gene expression analysis

At sacrifice, liver, skeletal muscle, and inguinal and epididymal adipose tissue were harvested, flash frozen, and RNA was extracted from ~100 mg of each tissue type for each animal using RNeasy mini-kits (Qiagen, Germantown, Maryland, USA). Generation of cDNA and quantitative real-time PCR were performed using Taqman probe sets (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [22]. Gene expression data were analyzed using the ΔΔCt method. Expression of 3 candidate housekeeping genes was measured for each tissue, and the genes exhibiting the most stable tissue-specific expression were utilized for data normalization. Gene expression was normalized to β-2-microglobulin (B2m) for liver, the geometric mean of B2m and non-POU-containing domain, octamer binding protein (NoNo) for adipose tissue, and β-actin (Actb) for skeletal muscle. See Table 1 for a complete list of Taqman primer sets used.

Table 1

Primer sets employed.

Gene name	Thermo Fisher Scientific accession number	Gene name	Thermo Fisher Scientific accession number
Acaca	Mm01304257_m1	Lep	Mm00434759_m1
Actb	Mm02619580_g1	Lpl	Mm00434770_m1
Adipoq	Mm00456425_m1	Nono	Mm00834875_g1
Atgl	Mm00503040_m1	Nrf1	Mm01135606_m1
B2m	Mm00437762_m1	Pck1	Mm01184322_m1
Ccl2	Mm00441242_m1	Pgk1	Mm00435617_m1
Cebpa	Mm00514283_s1	Ppargc1a	Mm00447184_g1
Cebpg	Mm01266786_m1	Ppara	Mm00440939_m1
Cpt1b	Mm00487200_m1	Pparg	Mm01184322_m1
Cyp19a1	Mm00484049_m1	Scd1	Mm00772290_m1
Dgat1	Mm00515643_m1	Slc2a4	Mm00436615_m1
Dgat2	Mm00499536_m1	Srebf1	Mm01163722_g1
Dlk1	Mm00494477_m1	Tfam	Mm00447485_m1
Emr1	Mm00802530_m1	Tgfb1	Mm01178829_m1
Esr1	Mm00433149_m1	Tnf	Mm00443258_m1
Hsl	Mm00495359_m1	Ucp1	Mm01244860_m1
Il6	Mm00446190_m1	Ucp2	Mm00627599_m1
Irs1	Mm01278327_m1

Plasma analyses and measurement of hepatic lipids

Plasma insulin, adiponectin, leptin, and IL-6 concentrations were measured at study weeks 8 and 24 (sacrifice) through commercially available ELISA kits (Millipore; Billerica, MA) according to the manufacturer’s instructions. Plasma 17β-estradiol concentrations were measured at week 24 (sacrifice) through ELISA (Calbiotech, Inc; El Cajon, CA). A modified Folch method was used to extract hepatic lipids [24], and colorimetric assays were used to measure hepatic cholesterol and triglyceride levels (Stanbio Laboratory; Boerne, TX) [22].

Quantification of 17β-estradiol and estrone concentrations within metabolic tissues

Tissue concentrations of estrone and 17β-estradiol were measured for skeletal muscle, liver, and epididymal adipose tissue. Inadequate sample remained for inguinal adipose tissue for these analyses. Estrogen concentrations were quantified through ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS-MS) as previously described in detail [25, 26]. The current analyses differed from prior methods only in that a Quatro-micro triple-quad mass spectrometer (Waters Corporation; Milton, MA) was employed. All other analytical parameters were similar to prior methods.

Statistical analyses

Unpaired Student’s t test was used to evaluate between-group differences at a single timepoint. Repeated measures ANOVA (RM-ANOVA) was used to determine whether an interaction between time and group was evident for study endpoints measured at more than 1 timepoint. Data are presented as mean ± standard deviation (SD) for cell culture data and mean ± standard error of the mean (SEM) for the remaining data. A p-value threshold of p<0.05 was used for statistical significance, and GraphPad Prism 5.0 software (GraphPad Software, Inc; La Jolla, CA) was used for figure creation and all statistical analyses.

Results

Peritoneal immune cells from male mice secrete estrogens

LC-MS/MS was used to quantify estrogen secretion from peritoneal immune cells to better define patterns of estrogen production. Thioglycolate-elicited peritoneal immune cells were harvested from male mice and cultured overnight. Steroid measurement demonstrated marked generation of 17β-estradiol (1667±295 pmol/L in conditioned media vs. <37 pmol/L in cell-free media; p<0.0001) and lesser production of estrone (196±28 pmol/L in conditioned media vs. 18±41 pmol/L in cell-free media; p<0.0001) (Fig 1B).

Aromatase deficiency in bone marrow-derived cells does not alter body weight or body composition in male mice

To verify successful engraftment of transplanted bone marrow, whole blood was collected 8 weeks post-transplant, and genotyping was performed on circulating white blood cells (Fig 2A). No differences in absolute or change in body weight (Fig 2B and 2C) were evident between WT(WT) and WT(ArKO) mice over 24 weeks on a regular chow diet, and body weights in both groups were nearly identical at sacrifice (26.4±0.5 gm in WT(WT) mice versus 26.5±0.5 gm in WT(ArKO) mice, p = 0.91).

Fig 2

Verification of bone marrow engraftment and body weight in wild-type (WT) male mice transplanted with bone marrow from either WT (WT(WT)) or aromatase-deficient (WT(ArKO) donors.

Genotyping of circulating white blood cells was performed to verify successful bone marrow engraftment (A), with illustrative results from 10 animals shown. No differences between groups were found in either absolute body weight (B) or change in body weight (C) over the 24-week study.

At study week 8 (12 weeks after bone marrow transplant), WT(ArKO) mice had significantly greater total fat mass than WT(WT) mice (1.7±0.1 gm versus 1.4±0.1 gm, respectively, p = 0.04; Fig 3A), but this difference in fat mass was no longer evident at study week 16 (Fig 3B). No differences in lean mass were seen between groups at either timepoint. Adiposity was measured again through fat-water imaging at study week 22 to assess body fat distribution. No differences in subcutaneous, visceral, or total fat volume were found between groups (Fig 3C). At sacrifice, epididymal adipose tissue mass did not differ significantly between WT(WT) and WT(ArKO) mice (18±1 mg/gm body weight in WT(ArKO) mice versus 16±1 mg/gm body weight in WT(WT) mice, p = 0.09; Fig 3D), and no difference was found between groups in inguinal white adipose tissue mass.

Fig 3

Body composition in wild-type (WT) male mice transplanted with bone marrow from either WT (WT(WT)) or aromatase-deficient (WT(ArKO) donors.

WT(ArKO) mice exhibited greater total body fat mass at week 8 (A) but not week 16 (B). At week 22, fat mass distribution was similar between groups (C). At sacrifice, tissue weights were similar between groups, although a trend was found toward greater epididymal fat pad weight in WT(ArKO) mice relative to WT(WT) mice (D). Liver lipid content was comparable between groups whether quantified by cholesterol and triglyceride mass (E) or liver fat volume as measured by MRI (F). *p<0.05.

Liver weights were comparable between both groups (50±1 mg/gm body weight in WT(WT) mice versus 48±2 mg/gm body weight in WT(ArKO) mice, p = 0.50; Fig 3D). Further, liver triglyceride and cholesterol content did not differ between WT(WT) and WT(ArKO) mice (Fig 3E), nor did total liver fat volume differ between groups as quantified by fat-water imaging (Fig 3F).

Male mice with hematopoietic aromatase deficiency exhibit enhanced glucose tolerance

Glucose tolerance and insulin sensitivity were assessed in WT(WT) and WT(ArKO) mice at study weeks 8 and 16 (12 and 20 weeks after bone marrow transplant, respectively). At study week 8, blood glucose did not differ between groups at any single timepoint during the glucose tolerance test, nor was the glucose area under the curve (AUC) different between groups (AUCglucose 3685±543 in WT(WT) mice versus 4825±684 in WT(ArKO) mice, p = 0.23; Fig 4A). Similarly, insulin sensitivity was comparable between groups at study week 8 (Fig 4B). However, at study week 16, WT(ArKO) mice exhibited significantly lower excursions in blood glucose during the glucose tolerance test (Fig 4C), as well as a lower glucose AUC (AUCglucose 6061±929 in WT(WT) mice versus 3406±379 in WT(ArKO) mice, p = 0.01), indicative of enhanced glucose tolerance compared to WT(WT) mice. Again, no difference in insulin sensitivity was seen between groups at study week 16 (Fig 4D).

Fig 4

Glucose tolerance and insulin sensitivity in wild-type (WT) male mice transplanted with bone marrow from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) donors.

At study week 8, no differences were seen in either glucose tolerance (A) or insulin sensitivity (B) between groups. In contrast, at study week 16, glucose tolerance was enhanced in WT(ArKO) mice relative to WT(WT) mice (C). Insulin sensitivity remained comparable between groups (D).

Consistent with findings of comparable insulin sensitivity between WT(WT) and WT(ArKO) mice, fasting plasma concentrations of insulin and glucose were similar between groups at study week 8 and at sacrifice (study week 24, 28 weeks after bone marrow transplant; Fig 5A and 5B). No differences were found between groups in plasma concentrations of the adipokines leptin (Fig 5C) or adiponectin (Fig 5D), nor did plasma lipid concentrations differ between WT(WT) and WT(ArKO) mice (Fig 5E and 5F). Finally, plasma concentrations of 17β-estradiol were similar between groups at baseline, study week 8, and sacrifice (Fig 5G).

Fig 5

Plasma analytes in wild-type (WT) male mice transplanted with bone marrow from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) donors.

At both study week 8 and study week 24, no differences were found between groups in fasting plasma concentrations of insulin (A), glucose (B), leptin (C), or adiponectin (D). Fasting plasma cholesterol (E) and triglyceride (F) concentrations were similar between groups at both timepoints, and plasma concentrations of 17β-estradiol remained comparable between groups throughout the study (G).

Loss of aromatase in hematopoietic cells confers increased skeletal muscle expression of genes implicated in insulin signaling and mitochondrial function

Consistent with the differences found in systemic glucose tolerance between WT(WT) and WT(ArKO) mice, differences were found between groups in the expression of genes implicated in energy metabolism within skeletal muscle (Fig 6A). Skeletal muscle from WT(ArKO) mice exhibited higher mRNA expression of glucose transporter type 4 (GLUT4, Slc2a4; p = 0.02), consistent with the finding of enhanced systemic glucose disposal. Further, WT(ArKO) mice had higher mRNA expression of the mitochondrial genes peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α, Ppargc1a; p = 0.03) and nuclear transcription factor 1 (NRF1, Nrf1; p = 0.01), suggesting enhanced energy metabolism and mitochondrial biogenesis. Skeletal muscle from WT(ArKO) also showed lower expression of the lipogenic enzyme stearoyl CoA desaturase-1 (SCD1, Scd1; p = 0.03), suggesting reduced ectopic lipid deposition. In WT(ArKO) mice, mean expression of ERα (Esr1) was higher relative to WT(WT) mice, although this difference did not achieve statistical significance (p = 0.09). Collectively these results suggest improvement in glucose and lipid metabolism in skeletal muscle from mice with hematopoietic aromatase deficiency.

Fig 6

Gene expression in metabolic tissues from wild-type (WT) male mice transplanted with bone marrow from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) donors.

Marked differences in skeletal muscle mRNA expression were found between groups (A), but gene expression profiles in liver (B), epididymal adipose tissue (C), and inguinal adipose tissue (D) were comparable in WT(ArKO) and WT(WT) mice. *p<0.05.

In contrast, no differences in mRNA expression were found for any of the targeted genes in liver (Fig 6B), epididymal adipose tissue (Fig 6C) or inguinal adipose tissue (Fig 6D). Whereas a trend was seen toward higher ERα (Esr1) expression among WT(ArKO) mice in skeletal muscle, no differences in ERα mRNA expression were found between WT(ArKO) and WT(WT) mice in liver, epididymal adipose tissue, or inguinal adipose tissue. Among these metabolic tissues, aromatase (Cyp19a) expression was detectable only in epididymal adipose tissue, and expression levels did not differ between groups.

Adipose tissue estrogen concentrations and immune cell infiltration

In epididymal adipose tissue, both estrone and 17β-estradiol concentrations were similar between groups (Fig 7A). Similarly, in liver, no between-group differences were found in tissue estrone concentration (485±165 pg/gm in WT(WT) mice versus 257±59 pg/gm in WT(ArKO) mice, p = 0.18) or 17β-estradiol concentration (3205±546 pg/mL in WT(WT) mice versus 2338±199 pg/mL in WT(ArKO) mice, p = 0.13; Fig 7B). In skeletal muscle, tissue estrone concentrations were comparable between groups (5804±967 pg/gm in WT(WT) mice versus 4336±392 pg/gm in WT(ArKO) mice, p = 0.15), but tissue 17β-estradiol concentrations were ~30% lower in WT(ArKO) mice relative to WT(WT) controls, although the difference did not achieve significance (5489±660 pg/gm in WT(WT) mice versus 3836±599 pg/gm in WT(ArKO) mice, p = 0.08; Fig 7C). Notably, this lack of significance was driven by a single outlier in the WT(ArKO) group, with a value >2 standard deviations above the mean for the remainder of the group. When this mouse was excluded from the analysis, tissue 17β-estradiol concentrations were significantly different between groups (5489±660 pg/gm in WT(WT) mice versus 3507±544 pg/gm in WT(ArKO) mice, p = 0.03).

Fig 7

Tissue estrogen concentrations in wild-type (WT) male mice transplanted with bone marrow from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) donors.

Tissue concentrations of estrone and 17β-estradiol were similar in epididymal adipose tissue (A) and liver (B). However, in skeletal muscle, a trend toward a lower concentration of 17β-estradiol was evident in WT(ArKO) mice relative to WT(WT) mice (C). Some values appear missing due to overlapping points in the scatterplots.

Within adipose tissue, no differences between WT(WT) and WT(ArKO) mice were observed in the number of infiltrating antigen-presenting cells as quantified by positive Mac-2 staining. Thus, in both epididymal (Fig 8A) and inguinal (Fig 8B) adipose tissue, comparable numbers of antigen-presenting cells were found between groups whether quantified as a percentage of total area or as cell number per field. Adipocyte size did not differ between groups in either inguinal or epididymal adipose tissue (Fig 8C and 8D).

Fig 8

Adipose tissue histology in wild-type (WT) male mice transplanted with bone marrow from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) donors.

Infiltration of antigen-presenting cells was similar in WT(WT) and WT(ArKO) mice in both epididymal (A) and inguinal (B) fat. Below graphs are representative images of Mac-2 staining of epididymal and inguinal adipose tissue from 2 animals in each group. Adipocyte size was similar in WT(WT) and WT(ArKO) mice in both epididymal (C) and inguinal (D) adipose tissue depots.

Discussion

These findings demonstrate novel evidence that immune cells contribute to total estrogen generation in metabolic tissues and support the importance of immune cell-derived estrogens for regulating metabolic health in males. Surprisingly, given the protective roles played by 17β-estradiol in tissue glucose homeostasis, male mice with selective aromatase deficiency in hematopoietic cells developed improved glucose tolerance relative to controls by 20 weeks after bone marrow transplant. This enhanced glucose tolerance occurred in the absence of differences in body weight, lean body mass, or adiposity between WT(WT) and WT(ArKO) mice. In parallel with enhanced systemic glucose tolerance, WT(ArKO) mice exhibited differential gene expression within skeletal muscle remarkable for greater expression of genes implicated in insulin signaling and mitochondrial function. Importantly, despite a lower tissue concentration of 17β-estradiol, WT(ArKO) mice also exhibited increased mRNA expression of ERα (Esr1) in skeletal muscle, indicating that loss of aromatase activity in hematopoietic cells may have led to a paradoxical increase in tissue-specific ERα signaling. Consistent with this idea, skeletal muscle exhibited enhanced expression of genes known to be positively regulated by 17β-estradiol. Interestingly, this phenomenon was present only in skeletal muscle, whereas adipose tissue and liver showed comparable estrogen concentrations and ERα mRNA expression in both groups. Importantly, however, these results are specific to a regular chow diet and may vary considerably in the setting of obesity, high-fat feeding, or other metabolic stressors. Collectively, these results implicate aromatase activity in immune cells particularly in skeletal muscle energy metabolism and underscore the need to better understand the intracrine and paracrine mechanisms through which immune cell-derived estrogens contribute to metabolic regulation in males.

Our study demonstrated reduced skeletal muscle 17β-estradiol concentrations but enhanced glucose tolerance in male mice with hematopoietic aromatase deficiency. This finding is in clear contrast to prior studies demonstrating impaired glucose tolerance in male mice with global aromatase deficiency or in female mice with loss of estrogen signaling in skeletal muscle conferred by ERα deficiency in either myocytes or myeloid cells [27-29]. Notably, however, tissue analyses also demonstrated higher ERα mRNA expression in skeletal muscle from WT(ArKO) mice, suggesting the possibility that selective loss of aromatase activity in immune cells yielded a compensatory increase in total estradiol signaling within skeletal muscle. A compensatory increase in estradiol signaling is further supported by the fact that skeletal muscle from WT(ArKO) mice showed enhanced expression of genes that are specifically upregulated by 17β-estradiol, including those involved in mitochondrial biogenesis [30]. Further, systemic administration of 17β-estradiol to orchiectomized mice enhanced skeletal muscle glucose uptake due to upregulation of GLUT4 expression [31], another gene that exhibited increased expression in the WT(ArKO) mice relative to WT(WT) controls. An alternative explanation for our findings is that the tissue-specific metabolic effects of estrogens are contingent on the cell of origin; thus, immune cell-derived estrogens could inhibit insulin signaling and mitochondrial biogenesis, and the protective metabolic effects of 17β-estradiol may derive exclusively from aromatase activity outside of the hematopoietic compartment.

Immune cells are resident in skeletal muscle, although they constitute well under 1% of total cells in muscle tissue [32]; these findings therefore are remarkable in that they suggest that immune cells are a key source of tissue estrogen generation and, despite their small numbers, contribute substantially to both total tissue estradiol concentrations and estradiol-mediated regulation of insulin sensitivity and mitochondrial function in skeletal muscle. Notably, although tissue estradiol concentrations differed by roughly 1/3, this finding did not achieve statistical significance and requires verification in a future study powered for this endpoint. As macrophages and lymphocytes are both present within skeletal muscle, additional work is needed to determine the respective contribution of each of these cell types to the observed differences in tissue estradiol concentrations, tissue-specific energy metabolism, and systemic glucose tolerance.

Previously, male mice with global aromatase deficiency exhibited greater body weight than WT controls by 3 months of age [27], although this body weight phenotype was not reproduced in a later study [33]. In the latter study, global aromatase deficiency resulted only in increased gonadal fat mass. In our study, hematopoietic aromatase deficiency did not yield lasting differences in body weight or total adiposity, but a trend toward higher epididymal fat mass was found in WT(ArKO) mice, suggesting that immune cell-specific aromatase deficiency may play a very minor, contributory role in the metabolic phenotype of global knockout mice. Nonetheless, loss of hematopoietic aromatase did not substantially affect adiposity in male mice. Similar body fat mass between WT(WT) and WT(ArKO) mice likely explains the comparable concentrations of circulating adipokines in both groups, in contrast to prior findings of elevated plasma leptin and reduced adiponectin concentrations in male mice with global aromatase deficiency [33]. One possible explanation for these overall null findings is that aromatase expression is high within adipose tissue, with aromatase activity in multiple cell types. Further, estrogens are stored in adipose tissue in the form of fatty acyl esters and estrogen sulfates and can be converted into bioactive estrogens through the activity of lipases and steroid sulfatase, respectively. It is possible, therefore, that estrogen generation through these alternative mechanisms fully compensated for loss of aromatase activity in tissue immune cells. This is particularly plausible as the animals were exposed only to a chow diet, so adipose tissue immune cell infiltration remained low. The metabolic importance of total aromatase activity within adipose tissue is supported by a recent study demonstrating that adipose-specific aromatase overexpression resulted in improved insulin sensitivity [34].

Further, WT(ArKO) mice did not develop time-dependent insulin resistance or hepatic steatosis, as has been observed in male mice with global aromatase deficiency and ascribed specifically to hepatic insulin resistance [27, 33]. Thus, in male mice with global loss of aromatase activity, overt hyperglycemia was seen by 3 months of age in association with increased expression of gluconeogenic genes in liver and, subsequently, increased hepatic triglyceride accumulation [33]. The present study did not reveal any evidence of changes in hepatic energy metabolism or insulin sensitivity, in contrast to these models of global aromatase deficiency in male mice. Therefore, hematopoietic aromatase does not appear to contribute to the hepatic insulin resistance or steatosis evident in global aromatase deficiency. Nonetheless, similar to findings in adipose tissue, we cannot definitively conclude whether the absence of a liver phenotype in the present study is due to a limited physiologic role for aromatase activity in liver immune cells or compensation through estradiol production by alternative pathways.

Plasma concentrations of 17β-estradiol were comparable between WT(WT) and WT(ArKO) mice, indicating that immune cells do not contribute significantly to circulating estrogens, at least via aromatization. Our findings further are consistent with prior work demonstrating similar plasma concentrations of triglycerides and cholesterol in mice with global aromatase deficiency and controls [27], though, in the prior study, global aromatase deficiency led to post-prandial hyperlipidemia after 36 weeks of age.

Our study has several key limitations. Although successful bone marrow engraftment was demonstrated in circulating immune cells, we cannot exclude the possibility that some host tissue immune cell populations persisted or expanded subsequent to transplant. However, unless the transplanted donor cells are expected to have impaired maturation or function, these donor cells should completely replace host tissue immune cells within 2–4 months after transplant in all metabolic tissues except brain [35]. Future work using labeled bone marrow cells will be important for careful quantitation of transplanted cells in metabolic tissues. Protein analyses also will be important to support the gene expression findings in our study; protein analyses were attempted for this cohort of animals but were not successful due to the methods employed for tissue processing at animal sacrifice. Future work also should include careful assessment of energy expenditure and food intake through calorimetry, particularly as body weight gain in male mice with global aromatase deficiency has been attributed at least in part to reduced volitional activity [9]. As noted previously, since estrogens can be generated within tissues through various pathways, our study cannot comprehensively define the metabolic roles of immune cell-derived estrogens; rather, it only addresses loss of estrogen production specifically through an aromatase-dependent mechanism. Therefore, future studies also should entail measurement of estrogen production through the steroid sulfatase pathway. Our study entailed radiation exposure and bone marrow transplant, which itself confers metabolic effects specifically with regard to reducing body weight gain, inhibiting adipogenesis, and increasing insulin resistance [36, 37]. However, these effects have been demonstrated with high fat feeding to confer nutritional stress, whereas our study employed a regular chow diet [36]. Critically, bone marrow transplant and ionizing radiation do not have a sustained impact on glucose homeostasis in mice exposed to a chow diet [36, 37], suggesting that our primary findings were not confounded by the use of this procedure. Exposure to ionizing radiation also was shown to inhibit proliferation in skeletal muscle satellite cells, although this was not associated with changes in insulin-mediated AKT phosphorylation within skeletal muscle [37]. Nonetheless, the possibility exists that employing bone marrow transplant contributed to our null findings in adipose tissue. Additional work is also needed to determine whether the observed differences in skeletal muscle energy metabolism are due to 17β-estradiol signaling or ligand-independent effects mediated by ERα. Growth factors, cytokines, and other signals have been shown to stimulate unliganded ERα-mediated transcriptional activation and repression [38-40], most commonly through serine phosphorylation [39]. Therefore, changes in ligand-independent ERα function may have contributed to the current findings. Finally, parallel work must be performed in models of high fat feeding and obesity to determine the interaction between metabolic stress and immune cell aromatase activity, as well as in female mice to establish whether sexual dimorphisms exist in the metabolic roles of immune cell-derived estrogens. Importantly, the chow diet employed in our study did not contain any ingredients that would contribute isoflavones or other phytoestrogens, as these have estrogenic properties and can achieve high circulating concentrations when administered through rodent diets [41].

Immune cells have extensive capacity to synthesize and secrete estrogens. These findings demonstrate initial evidence that this facet of immune cell function may play important metabolic roles. Extensive additional studies are needed to delineate the respective intracrine and paracrine effects of immune cell-derived estrogens and to determine how immune cell aromatase activity is regulated within metabolic tissues. Finally, this study contributes to a growing body of evidence demonstrating the importance of estrogens for influencing metabolic health in males.

29 Oct 2019

PONE-D-19-24315

Aromatase deficiency in hematopoietic cells improves glucose tolerance in male mice through skeletal muscle-specific effects

PLOS ONE

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The study by Rubinow et al attempts to investigate the role of estradiol produced by immune cells in regulating metabolism. To achieve this, the authors use a clever approach of performing bone marrow transplantation experiments in mice using WT and aromatase-deficient donors.

The authors clearly demonstrate that cultured immune cells isolated from the peritoneal of mice secrete high levels of 17B-Estradiol and estrone. The phenotype of the WT mice transplanted with bone marrow from ArKO mice however is mild with only statistically significant differences being identified as an increase in fat mass at 8 weeks and a decrease in glucose tolerance at 16 weeks of age.

Unfortunately, the authors have over-stated the data with the first 3 paragraphs of the discussion based on data that is not statistically significant, particularly with regards to the gene expression data in skeletal muscle and the concentrations of 17B-estradiol within skeletal muscle. Throughout the manuscript, the authors use the word “trend” to describe data with P values of >0.05 (eg. P=0.09).

Whilst the premise for this study has the potential to provide important insight into the role of estradiol derived from immune cells in metabolic regulation, the data presented in this paper is premature and requires additional analyses to ensure accurate interpretation of the data.

Other comments:

Why are the data expressed at Mean +/- SD in the text and SEM in the figures? These should be consistent between the text and the figures. SD is appropriate for replicates of cell culture experiments, and SE for experiments consisting of individual animals.

The gene expression data in skeletal muscle could be further supported by protein analyses.

The housekeeping genes Gapdh and Actb have been shown not to be optimal for normalisation of gene expression data as they are not stably expressed. As such, alternative house keeping genes should be used.

Reviewer #2: Estrogen is known to play an important metabolic role in females and have now been acknowledged to have a role in males. Similar to females, loss of estogen results in increased body mass due to increased fat mass, and reduced insulin sensitivity. Thus, having an overall negative effect on the metabolic health of the individual. Additionally, global aromatase deficiency results in a similar metabolic phenotype, but the mechansms for estrogen-based metabolic regulation in males is not completely understood. Therefore, the authors aim to uncover the source of estrogen production and tissue-specific site of action which results in these metabolic defects in males. Thus, the authors aim to examine the metabolic effects of estrogens specfically produced by immune cells. The authors emply bone-marrow transplants from wild-type and aromatase knock-out animals into wild-type animals in order address this question. While the results from this manuscript conflict the results from previous studies the authors do not over interpret their results and are transparent with their study limitations. Minor comments to improve the manuscript are as follows:

1. Please report more specific RT-qPCR methods such as (taqman or syber green): supplementary table with genes analyzed and primer sequences

2. While transcript data is useful, protein data would be more valuable and strengthen the data to support the conclusions that these advantageous metabolic adaptations occurred.

3. The addition of metabolic chamber data would strengthen the manuscript but not required.

**********

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12 Dec 2019

We thank the journal and reviewers for their critique of our paper. Please find below our itemized responses to the journal requirements and reviewers’ comments.

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We have reviewed all style requirements and labeled files as indicated.

2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

We apologize for use of this phrase. We have removed the phrase in reference to the food intake data (lines 257-258); as the animals were group housed, we only have estimates for food intake data for the individual animals. Although these estimates do not suggest a difference in food intake, we have chosen to omit this altogether in the absence of higher quality data.

This phrase also was used in reference to measurements of adipocyte size. These data are now shown in Figures 8C and 8D.

3. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: 1) Please provide the number and source of the donor mice 2) Please provide details of animal welfare for both the donor and recipient mice (e.g., shelter, food, water, environmental enrichment) 3) please describe any steps taken to minimize animal suffering and distress, such as by administering analgesics, for the recipient mice 4) please include the method of sacrifice for both the donor and recipient mice and 5) Please describe the post-operative care received by the recipient animals, including the frequency of monitoring and the criteria used to assess animal health and well-being. Thank you for your attention to these requests.

We apologize for omission of this information and have added the following details to our manuscript:

1) Number and source of donor mice: The breeder mice were from a congenic C57BL6/J strain and provided from an animal colony at the University of Texas-Southwestern maintained by the senior author (OKO); these included 1 male and 2 female mice heterozygous for aromatase deficiency and 1 wild-type male mouse. (lines 113-115)

2) Details of animal welfare: All mice were group housed (4-5 animals per cage, breeding pairs 2 animals per cage) with ad libitum access to food and water. (lines 176-177)

3) Steps to alleviate animal suffering: In order to minimize suffering and risk to animals, fasting times were limited to 4-5 hours prior to metabolic testing, bone marrow transplant and sacrifice were performed under isoflurane anesthesia, and mice were placed on heating pads immediately following fat-water imaging. Adequacy of anesthesia was assessed through monitoring of respiratory rate and toe pinch response. (lines 177-181)

4) Method of sacrifice:

Donor mice: The morning following irradiation, donor mice were sacrificed by CO2 inhalation and exsanguination, and femurs and tibias were harvested. (lines 143-144)

Recipient mice: Measurement of visceral, subcutaneous, and liver fat volume subsequently was performed using fat-water imaging (study week 22), and animals were sacrificed at study week 24 through cervical dislocation and exsanguination. (lines 172-174)

5) Post-operative care for recipient mice: Recipients of bone marrow transplant were monitored 3-4 times each week for the 4 weeks subsequent to irradiation until marrow engraftment occurred. Mice were monitored for any signs or behaviors indicative of poor health, including hunching, difficulty breathing, or listlessness, and any concerning signs or behaviors were reported to veterinary medicine. (lines 130-134)

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

________________________________________

Reviewer #1: The study by Rubinow et al attempts to investigate the role of estradiol produced by immune cells in regulating metabolism. To achieve this, the authors use a clever approach of performing bone marrow transplantation experiments in mice using WT and aromatase-deficient donors.

The authors clearly demonstrate that cultured immune cells isolated from the peritoneal of mice secrete high levels of 17B-Estradiol and estrone. The phenotype of the WT mice transplanted with bone marrow from ArKO mice however is mild with only statistically significant differences being identified as an increase in fat mass at 8 weeks and a decrease in glucose tolerance at 16 weeks of age.

Unfortunately, the authors have over-stated the data with the first 3 paragraphs of the discussion based on data that is not statistically significant, particularly with regards to the gene expression data in skeletal muscle and the concentrations of 17B-estradiol within skeletal muscle. Throughout the manuscript, the authors use the word “trend” to describe data with P values of >0.05 (eg. P=0.09).

We appreciate the reviewer’s approval of our approach. Although there was disagreement between reviewers regarding the stringency of our interpretation of the data, we appreciate these comments and did not intend to overstate the findings. We agree the phenotype is mild but nonetheless think the results are strengthened by the consistency between the enhancement of systemic glucose tolerance and gene expression data in skeletal muscle in the WT(ArKO) group. Additionally, we believe the biological significance of our findings is supported by the fact that changes were observed in skeletal muscle but not liver or adipose. To better avoid overstating our findings, we have made the following revisions:

We have changed the text in our Results section to state that epididymal adipose tissue mass did not differ significantly between groups (lines 272-275), particularly as this finding was found only on post-mortem tissue weight analysis and not corroborated by our fat-water imaging data.

We also have modified our reporting of gene expression data to present p-values and focus solely on the genes that differed significantly between groups with the exception of Esr1, which we think may be important for interpreting the results collectively. These changes have been made to Abstract (lines 31-32) and the Results section, where mention of the genes Irs1, Tfam, and Cptb1b have been removed from the text (lines 332-342).

With regard to the skeletal muscle concentrations of 17β-estradiol, we now have presented the figures as scatterplots, in order to better depict that much of the variation in tissue concentrations in the WT(ArKO) group was driven by a single outlier. Although some choose to exclude outliers >2 SD from the mean, we opted instead to show all data but add a sensitivity analysis demonstrating a significant between-group difference with exclusion of this single outlier (lines 369-373). Nonetheless, as this outlier did introduce substantial variation into the data, we explicitly state that the between-group difference did not achieve statistical significance (line 368), and we reiterate in the discussion that the between-group difference in skeletal muscle estradiol concentrations did not achieve significance when all animals were included in the analysis (lines 439-441):

Notably, although tissue estradiol concentrations differed by roughly 1/3, this finding did not quite achieve statistical significance and requires verification in a future study powered for this endpoint.

Whilst the premise for this study has the potential to provide important insight into the role of estradiol derived from immune cells in metabolic regulation, the data presented in this paper is premature and requires additional analyses to ensure accurate interpretation of the data.

We think the data are not premature as related to sample size and the publication philosophy of the journal. Rather, the data reflect the magnitude of biological effects in our experimental design. We have rephrased our interpretation toward a much more conservative interpretation. Further, we think our studies were carried out in a technically sound way, a requirement of the journal.

Other comments:

Why are the data expressed at Mean +/- SD in the text and SEM in the figures? These should be consistent between the text and the figures. SD is appropriate for replicates of cell culture experiments, and SE for experiments consisting of individual animals.

We appreciate this suggestion. Cell culture data are now shown as mean ± SD in the figure as well as in the text, and all other data are now shown as mean ± SEM. The statistical analysis section of the Methods has been updated accordingly (lines 238-240).

The gene expression data in skeletal muscle could be further supported by protein analyses.

We agree that protein analyses are important for supporting our gene expression data. Consequently, we extracted protein from skeletal muscle and performed Western blotting for ERα, aromatase, and GLUT4, as well as β-tubulin and β-actin for normalization. Unfortunately, we only had formalin-fixed tissue remaining, and despite the use of 2 different protein extraction methods, we failed to get a signal for any of the probed proteins, including β-tubulin and β-actin. We very much regret that we are unable to include these additional protein analyses in our paper.

The housekeeping genes Gapdh and Actb have been shown not to be optimal for normalisation of gene expression data as they are not stably expressed. As such, alternative house keeping genes should be used.

Thank you for this suggestion. Gene expression in epididymal and inguinal adipose tissue has been re-analyzed using the geometric mean of Nono and β-2-microglobulin (B2m) rather than Gapdh for normalization. For the skeletal muscle gene expression data, additional genes for normalization were analyzed, including B2m and 18s. However, when all of the normalization genes were compared, β-actin demonstrated the most stable expression. Therefore, our further work to identify an optimal gene for normalization supported continued use of β-actin as our housekeeping gene for this tissue.

Reviewer #2: Estrogen is known to play an important metabolic role in females and have now been acknowledged to have a role in males. Similar to females, loss of estrogen results in increased body mass due to increased fat mass, and reduced insulin sensitivity. Thus, having an overall negative effect on the metabolic health of the individual. Additionally, global aromatase deficiency results in a similar metabolic phenotype, but the mechanisms for estrogen-based metabolic regulation in males is not completely understood. Therefore, the authors aim to uncover the source of estrogen production and tissue-specific site of action which results in these metabolic defects in males. Thus, the authors aim to examine the metabolic effects of estrogens specifically produced by immune cells. The authors employ bone-marrow transplants from wild-type and aromatase knock-out animals into wild-type animals in order address this question. While the results from this manuscript conflict the results from previous studies the authors do not over interpret their results and are transparent with their study limitations. Minor comments to improve the manuscript are as follows:

1. Please report more specific RT-qPCR methods such as (taqman or syber green): supplementary table with genes analyzed and primer sequences.

We appreciate this suggestion. Greater detail regarding the PCR methods employed has been added to the Methods section (lines 206-214), and Table 1 has been inserted (line 215) and lists the accession numbers for all primer sets used in the analyses.

2. While transcript data is useful, protein data would be more valuable and strengthen the data to support the conclusions that these advantageous metabolic adaptations occurred.

We agree with this reviewer and have performed additional protein analyses as described above in response to Reviewer #1. Despite multiple attempts to extract protein from our formalin-fixed tissues, we are unable to provide protein data for the manuscript.

3. The addition of metabolic chamber data would strengthen the manuscript but not required.

We also agree with this suggestion. Due to limited funding, we were unable to include metabolic chamber analyses in this study. Although we did not observe a body weight or body composition phenotype in our WT(ArKO) animals, we agree that metabolic chamber data will be necessary in a future study to comprehensively define the metabolic phenotype of mice with hematopoietic aromatase deficiency.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

19 Dec 2019

PONE-D-19-24315R1

Aromatase deficiency in hematopoietic cells improves glucose tolerance in male mice through skeletal muscle-specific effects

PLOS ONE

Dear Dr. Rubinow,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points still raised by reviewer 1.

We would appreciate receiving your revised manuscript by Feb 02 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Michael Bader

Academic Editor

PLOS ONE

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1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

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Reviewer #1: Yes

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6. Review Comments to the Author

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Reviewer #1: The authors have attempted to address each of the editor’s and reviewer’s concerns and is greatly improved as a result, particularly with regards to the over-interpretation of non-significant data in the first version of the manuscript.

Minor comments;

It is worth noting in the text, that the skeletal muscle gene expression data was expressed relative to beta-actin as it was demonstrated to be the most stably expressed housekeeping gene of those analysed.

It is also worth acknowledging in the text that the gene expression data in skeletal muscle could be further supported by protein analyses, however, this was not possible using the current cohort of samples due to the method in which the tissues were processed.

Line 342 – The use of the word “trend” should be avoided to describe data with a P value of P=0.09. The description of these data could be re-worded to state that the mean value of ERalpha mRNA was higher in the WT(ArKO) mice than the WT mice however this did not reach statistical significance (P=0.09).

Lines 367-368 and Lines 439-441 – Similarly, this sentence should be reworded to state that the mean 17B estradiol levels were lower in WT(ArKO) mice, but this did not reach statistical significance P=0.08. The word “quite” in this sentence implies a P value close to 0.05 (ie. such as 0.06), so should be removed.

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Reviewer #1: No

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27 Dec 2019

We thank the reviewer for additional suggestions for our paper. Please find below our itemized responses to the reviewer’s comments.

Comments to the Author

________________________________________

Reviewer #1: The authors have attempted to address each of the editor’s and reviewer’s concerns and is greatly improved as a result, particularly with regards to the over-interpretation of non-significant data in the first version of the manuscript.

Minor comments;

It is worth noting in the text, that the skeletal muscle gene expression data was expressed relative to beta-actin as it was demonstrated to be the most stably expressed housekeeping gene of those analysed.

We agree that this additional information is important to include and have added the following sentence to our Methods section (lines 209-211):

Expression of 3 candidate housekeeping genes was measured for each tissue, and the genes exhibiting the most stable tissue-specific expression were utilized for data normalization.

It is also worth acknowledging in the text that the gene expression data in skeletal muscle could be further supported by protein analyses, however, this was not possible using the current cohort of samples due to the method in which the tissues were processed.

We now include this limitation of our study in our discussion, as follows (lines 480-482):

Protein analyses also will be important to support the gene expression findings in our study; protein analyses were attempted for this cohort of animals but were not successful due to the methods employed for tissue processing at animal sacrifice.

Line 342 – The use of the word “trend” should be avoided to describe data with a P value of P=0.09. The description of these data could be re-worded to state that the mean value of ERalpha mRNA was higher in the WT(ArKO) mice than the WT mice however this did not reach statistical significance (P=0.09).

We appreciate this comment, and the wording of this sentence has been amended as suggested (lines 336-338):

In WT(ArKO) mice, mean expression of ERα (Esr1) was higher relative to WT(WT) mice, although this difference did not achieve statistical significance (p=0.09).

Lines 367-368 and Lines 439-441 – Similarly, this sentence should be reworded to state that the mean 17B estradiol levels were lower in WT(ArKO) mice, but this did not reach statistical significance P=0.08. The word “quite” in this sentence implies a P value close to 0.05 (ie. such as 0.06), so should be removed.

The word ‘quite’ has been removed in both of these places, as advised (lines 362 and 434).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

31 Dec 2019

Aromatase deficiency in hematopoietic cells improves glucose tolerance in male mice through skeletal muscle-specific effects

PONE-D-19-24315R2

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Additional Editor Comments (optional):

Reviewers' comments:

6 Jan 2020

PONE-D-19-24315R2

Aromatase deficiency in hematopoietic cells improves glucose tolerance in male mice through skeletal muscle-specific effects

Dear Dr. Rubinow:

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