
==== Front
Mol Metab
Mol Metab
Molecular Metabolism
2212-8778
Elsevier

S2212-8778(24)00139-X
10.1016/j.molmet.2024.102008
102008
Original Article
Glucose intolerance as a consequence of hematopoietic stem cell dysfunction in offspring of obese mice
Denizli Merve 18
Ropa James 238
Beasley Lindsay 2
Ghosh Joydeep 4
DeVanna Kelli 1
Spice Taylor 1
Haneline Laura S. 125
Capitano Maegan malcapit@iu.edu
2⁎⁎
Kua Kok Lim kkua@iu.edu
1567⁎
1 Department of Pediatrics, Neonatal-Perinatal Medicine, Indiana University School of Medicine, Indianapolis, 46202, USA
2 Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, 46202, USA
3 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, 46202, USA
4 Department of Medicine, Indiana University School of Medicine, Indianapolis, 46202, USA
5 Department of Anatomy, Cell Biology & Physiology, Indiana University School of Medicine, Indianapolis, 46202, USA
6 Center for Diabetes and Metabolic Disease, Indiana University School of Medicine, Indianapolis, 46202, USA
7 Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, 46202, USA
⁎ Corresponding author. kkua@iu.edu
⁎⁎ Corresponding author. malcapit@iu.edu
8 These authors contributed equally to the work as first authors.

12 8 2024
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© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Objective

Maternal obesity is increasingly common and negatively impacts offspring health. Children of mothers with obesity are at higher risk of developing diseases linked to hematopoietic system abnormalities and metabolism such as type 2 diabetes. Interestingly, disease risks are often dependent on the offspring's sex, suggesting sex-specific reprogramming effect of maternal obesity on offspring hematopoietic stem and progenitor cell (HSPC) function. However, the impact of maternal obesity exposure on offspring HSPC function, and the capability of HSPC to regulate offspring metabolic health is largely understudied. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health.

Methods

We first assessed bone marrow hematopoietic stem and progenitor cell phenotype using postnatal day 21 (P21) and 8-week-old C57BL/6J mice born to control and diet-induced obese dams. We also sorted HSPC (Lineage-, Sca1+, cKit + cells) from P21 mice for competitive primary and secondary transplant, as well as transcriptomic analysis. Body weight, adiposity, insulin tolerance test and glucose tolerance tests were performed in primary and secondary transplant recipient animals.

Results

We discovered sex-differences in offspring HSPC function in response to maternal obesity exposure, where male offspring of obese dams (MatOb) showed decreased HSPC numbers and engraftment, while female MatOb offspring remained largely unaffected. RNA-seq revealed immune stimulatory pathways in female MatOb offspring. Finally, only recipients of male MatOb offspring HSPC exhibited glucose intolerance.

Conclusions

This study demonstrated the lasting effect of maternal obesity exposure on offspring HSPC function and implicates HSPC in metabolic regulation.

Graphical abstract

Image 1

Highlights

• Maternal obesity induces sex-specific changes in offspring hematopoietic stem and progenitor cell (HSPC).

• HSPCs from male offspring of obese mice had reduced engraftment during transplantation, while female HSPCs were unaffected.

• Recipient animals mirrored glucose intolerance seen in donor offspring, highlighting HSPC's role in glucose metabolism

Keywords

Hematopoietic stem cell
Offspring exposed to maternal obesity
Stem cell transplant
==== Body
pmc1 Introduction

The incidence of obesity [body mass index (BMI)≥30] is increasing in all age groups, including women of childbearing age [1,2]. In 2019, the CDC reported that over 29% of mothers had a BMI≥30 before and during pregnancy, with over one million infants born to mothers with obesity [3]. Epidemiological studies show that children born to mothers with obesity experience a higher risk of developing numerous chronic illnesses [[4], [5], [6]]. Specifically, human offspring exposed to maternal obesity were found to have higher risks of developing neuropsychiatric disorders, allergy/asthma, cardiometabolic diseases such as obesity and type 2 diabetes mellitus, and leukemia later in life [[4], [5], [6], [7], [8], [9]]. These observations are in line with the concept of developmental origins of health and diseases (DOHaD) [10] suggesting that alterations to the in-utero environment impact the development of the fetus and subsequently heighten the risk of chronic illnesses in adulthood.

To date, clinical studies have shown that offspring of mothers with obesity are more likely to develop obesity and suffer higher risks of developing chronic diseases such as non-alcoholic fatty liver disease [4], type 2 diabetes [11], and asthma [5]. Children born to mothers with obesity also experience higher hospitalization rates from infection [6,7]. Additionally, they face an increased risk of acute lymphoblastic leukemia [8,9]. Notably, these diseases share common features of immune dysfunction and altered hematopoiesis, highlighting the potential impact of maternal obesity exposure on offspring hematopoietic stem and progenitor cell (HSPC) function.

Animal offspring exposed to maternal obesity have recapitulated several disease phenotypes observed in human offspring of mothers with obesity. These models are widely used as a tool to define the impact of maternal obesity on different organ systems in offspring, as well as the mechanistic pathways underpinning offspring organ dysfunction [[12], [13], [14], [15], [16], [17], [18]]. Using a Western diet induced maternal obesity murine model, our group recently reported sex-differences in offspring glucose intolerance and pancreatic beta cell function [13]. Specifically, we found that both male and female offspring of obese dams developed increased body adiposity compared to same sex controls, but interestingly only male offspring of obese dams developed significant glucose intolerance and pancreatic beta cell dysfunction [13]. A similar murine model of diet-induced maternal obesity reported altered hematopoiesis [19]. Additionally, non-human primate studies have reported altered fetal HSPC function in offspring exposed to maternal obesity induced by Western diet, and this effect was observed until young adulthood [12,13,20]. Taken together, the animal studies also underscore the impact of maternal obesity exposure on offspring HSPC function.

Obesity-induced pro-inflammatory states play a vital role in the development of chronic illnesses in adults. Although the mechanistic pathways underpinning the relationship between obesity and inflammation have not been fully identified, elevated production of pro-inflammatory markers such as interleukin (IL)-6, IL-8, C-reactive protein (CRP), interferon (IFN)-γ, and tumor necrosis factor (TNF)-α as well as altered adipokines and gut microbiota are known to be associated with increased BMI [[21], [22], [23]]. Diet-induced obesity leads to suppressed HSPCs as well as an impaired immune response against infections in adults [24,25]. In line with this background, both human and animal offspring born to mothers with obesity are at risk of developing obesity; this further raises the potential of HSPC dysfunction in offspring. In fact, several studies have shown that maternal obesity exposure leads to changes in the landscape of immune cells in offspring [23,[26], [27], [28]]. However, the impact of maternal obesity on offspring HSPC function, particularly in response to prolonged stress, and its potential direct role in chronic illnesses in offspring, is largely understudied. Further, sex-differences in offspring HSPC function have not been well-characterized.

In this study, we deployed a murine model of maternal obesity that induces sex-specific glucose intolerance in offspring to define the sex-specific differences and factors contributing to altered HSPC function in murine offspring exposed to maternal obesity. The primary hypothesis of this study was that the hematopoietic transcriptome of offspring is affected by maternal diet in a sex-specific manner that ultimately leads to sex-differences in HSPC function. As we previously identified the sex-differences in glucose intolerance in offspring born to a murine model of diet-induced maternal obesity [13], we also hypothesized the role of HSPCs in regulating glucose tolerance in offspring of obese mice. We utilized competitive transplant assays and an unbiased transcriptomic study to elucidate sex-differences in altered HSPC function in offspring exposed to maternal obesity, as well as the novel role of HSPCs in regulating total body glucose tolerance.

2 Materials and methods

2.1 Animals

All procedures adhered to the regulations of the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee. Animals were housed in a temperature-controlled, 12-hour light–dark cycled animal care facility with free access to water and food. For the maternal obesity model C57BL/6J female mice were purchased from Jackson Laboratory (Bar Harbor, ME). For the transplantation analysis, 8–10 week old Boy/J, and B6 Boy/J F1 (referred to as F1) mice were obtained from an on-site breeding core facility at the Indiana University School of Medicine.

2.2 Diet induced maternal obesity

Four to five-week-old C57BL/6J female mice acclimatized to the animal facility for >72 h prior to randomization. Female dams were randomly assigned to regular chow (2018SX; Inotiv, IL) as a control, or Western diet (TD.88,137; Inotiv, IL) to induce maternal obesity as published previously [13]. The dams received the same diet for four weeks prior to mating, and through pregnancy as well as during lactating period (Figure 1A). After birth, each litter was culled to maximum of 8 pups. On postnatal day 21 (P21), offspring were euthanized for bone marrow (BM) extraction or weaned to a regular chow diet and evaluated at 8-weeks (8W). 8-week-old female offspring were not exposed to males upon weaning, and the estrous cycle were not synchronized/evaluated prior to metabolic studies to minimize stress.Figure 1 Maternal obesity model. (A) Female mice were fed regular chow (Con) or Western diet (TD 88137) (MatOb) 4 weeks prior to mating, through pregnancy and lactating period. Offspring were weaned to regular chow diet at P21. (B–J) Evaluation at P21. Maternal obesity causes a significant decrease in (B) LT-HSC and (C) ST-HSC cells in male MatOb offspring (n = 7–8 mice from 4 litters/sex/group). (D) CLP, (E) CMP, (G) MEP is unchanged between groups while (F) GMP is significantly decreased in male MatOb offspring (n = 6–8/group). (H–J) Evaluation of mature hematopoietic cells on P21 showed decrease in myeloid cells in female and reduced CD3+ cells in both sexes, no changes were detected in B220 cells (n = 4 mice from 2 litters/sex/group). (K–L).

Figure 1

2.3 Immunophenotyping

Offspring born to chow-fed control dams and diet-induced obese dams were euthanized on P21 and BM was obtained from femurs, tibiae, and hips. When required, to enhance the rate of cell sorting using flow cytometry, lineage depletion was performed using the EasySep™ Mouse Hematopoietic Progenitor Cell Isolation kit (STEMCELL Technologies). Details for antibodies and definitions of HSPC populations are included in the Supplemental Methods.

2.4 Competitive primary transplant assay

Donor BM from two C57Bl/6J (CD45.2+ CD45.1-) P21 offspring were pooled and stained for fluorescence activated cell sorting (FACS) to isolate LSK (Lin- Sca1+ c-Kit1+) cells directly into RPMI-1640 containing 10% FBS. Each pool of 1000 freshly-sorted LSK were injected intravenously with 200,000 unseparated Boy/J (CD45.1+ CD45.2-) BM cells into two to three lethally irradiated (700 cGy followed with 400 cGy) F1 recipient (CD45.1+ CD45.2+) mice. Donor chimerism (%CD45.1- CD45.2+) was determined in peripheral blood (PB) at week 4 and 16, and in BM at week 16.

2.5 Secondary transplant assay

After week 16 post transplantation, primary recipient mice were euthanized, and BM harvested. Recipient mouse BM from the same group were pooled at equal cells/mouse ratios. One million primary recipient BM cells were transplanted into lethally irradiated F1 recipients. Donor chimerism (%CD45.1- CD45.2+) was determined in PB at week 4 and 16, and BM at week 16 post-transplant.

2.6 RNA sequencing workflow and analysis

Details of RNA sequencing workflow and analysis are included in Supplemental Methods. Briefly, BM LSK cells from P21 offspring were collected by FACS into lysis buffer (n = 6/sex/group). RNA was harvested (Qiagen RNeasy Micro Plus Kit). mRNA-seq libraries were prepared using samples with RIN >8.0 with NEBNext Ultra Low Input/Single Cell RNA sequencing library preparation kit. Paired end sequencing was performed using an Illumina NovaSeq v1.5 S4 sequencer. Reads were aligned using STAR sequence aligner and DESeq2 was used for differential gene expression analysis using the design ∼ Batch + Diet for comparison of MatOb compared to control controlling for litter and different dates of collection within each sex group, with adjusted p-value of 0.05. The R package “fgsea” was used to perform gene set analysis using the MSigDB curated datasets (https://bioinf.wehi.edu.au/software/MSigDB/). RNA-seq raw and processed data files are deposited to Gene Expression Omnibus (GEO) (GSE253870).

2.7 Cytokine analysis

Serum was collected from non-fasting P21 and 8W offspring. IFN-α, IFN-β, IFN-γ, IL-10 were measured using MSD U-Plex platform (Meso Scale Diagnostics, Rockville, Maryland). Serum IL-10 in primary transplant recipient animals was measured using Murine IL-10 Standard TMB ELISA Development Kit (Peprotech, Cranbury, New Jersey) following manufacturer's instructions.

2.8 In vivo metabolic evaluations of recipient animals

The body composition of primary and secondary transplant recipients was assessed 16 weeks post transplantation using EchoMRI (EchoMRI LLC, Houston, TX). Intraperitoneal glucose tolerance testing (GTT) and intraperitoneal insulin tolerance testing (ITT) were performed as published previously [13]. Additional details are provided Supplemental Methods.

2.9 Immunohistochemistry and β-cell area measurement

Pancreata of recipients were fixed and sectioned at the thickness of 5 μm slices. Two to four sections per animal were analyzed for β-cell area [13,29]. Pancreatic sections were processed, stained, and analyzed as previously described. Immunohistochemistry was performed using rabbit anti-insulin antibody (#3014, Cell Signaling). Images of whole pancreas were obtained using Axio-Scan Z1 inverted microscope, and the percent area of β-cell was calculated by dividing insulin-stained area with total pancreatic area as published [13,29].

2.10 Statistics

Statistical analysis was performed using GraphPad Prism 10 unless otherwise indicated. Details are provided in the Supplemental Methods.

3 Results

3.1 Maternal obesity exposure causes phenotypic changes in offspring HSPCs

We first assessed the HSPC subsets in bone marrow (BM) of offspring on postnatal day 21 (P21) and at 8-weeks old (8 W) to define the sex-specific impact of maternal obesity (MatOb) exposure on offspring hematopoiesis (Figure 1A). At P21, male MatOb offspring had a significant reduction in frequency of both long-term hematopoietic stem cells (LT-HSCs) and short-term hematopoietic stem cells (ST-HSCs) (Figure 1B, C) compared to same-sex controls (Con). In contrast, P21 female MatOb pups were not significantly different from same-sex Con. Common lymphoid progenitor (CLP), common myeloid progenitor (CMP), and megakaryocyte-erythroid progenitor cells (MEP) were unchanged by MatOb exposure regardless of sex (Figure 1D, E and 1G). Compared to same-sex Con, there was a significant decrease in granulocyte-macrophage progenitors (GMP) in male MatOb offspring, whereas GMP were unchanged in female MatOb offspring (Figure 1F). Female MatOb offspring had significantly lower frequencies of Gr1+Mac1+ mature myeloid cells (Figure 1H). Both male and female MatOb offspring had a significant decrease in CD3+ T-cell numbers (Figure 1I). In contrast, B220+ B-cell numbers remained unchanged compared to same-sex Con (Figure 1J). In summary at P21, there were sex-specific alterations in HSPCs of offspring mice exposed to maternal obesity compared to control.

3.2 Transcriptomic analyses reveal inflammatory gene pathways in female MatOb offspring HSPCs

We next isolated LSK cells, which are enriched for HSCs and multipotent progenitor cells, for RNA-seq from MatOb or Con offspring to assess differentially expressed genes and gene programs that are enriched in response to maternal obesity exposure. There were 100 differentially expressed genes between MatOb and Con in males (Figure 2A, Supplemental Table 1) and 1788 differentially expressed genes in females (Figure 2B, Supplemental Table 2). Interestingly, there were more and higher magnitude of differential expression in female mice than male. 72% of the genes differentially expressed in male pups are also differentially expressed in females (Figure 2C, D). This includes many of the top differentially expressed genes altered by maternal diet in both male and female pups. These include genes such as Car1 and Spta1, which are associated with erythroid development and are significantly enriched in Con compared to MatOb offspring in both male and female (Figure 2E, F). Cpt1a and Scd2, which are associated with metabolic processes, are significantly enriched in male MatOb offspring compared to same-sex Con (Figure. 2G). There are also many genes highly differentially expressed in female pups that are unaltered in male pups. Slc30a10, which plays a role in metal transport, is significantly enriched in Con compared to MatOb only in female offspring (Figure 2F). Interestingly, Serpina3g, which plays a role in HSPC post-stress recovery, is enriched only in female MatOb offspring compared to Con (Figure 2H).Figure 2 RNA was harvested from Lin-Sca1+Kit + bone marrow cells from P21 MatOb or Control pups and subjected to RNA-sequencing. (A–B) Plots showing the average normalized read counts vs the fold-change (FC) of MatOb/Control (MA plots) for every detected gene for male (A) and female (B) offspring. Red dots indicate genes significantly enriched in MatOb while blue dots indicate genes significantly enriched in control. (C–D) Venn diagrams showing overlap between male and female pups of genes that are differentially enriched in control (C) or in MatOb (D), separated by sex-matched samples. Orange circle on left represents DEG in female MatOb offspring LSK, while dark green circle on right represents DEG in male MatOb offspring LSK. (E–H) Top genes in male (E/G) and female (F/H) pups that are significantly differentially enriched in control (E–F) or in MatOb (G–H). Bolded font represented DEG with adjusted p-value<0.05. n = 6 animals from 6 separate litters/sex/group. Significantly differentially expressed genes were assessed using DESeq2 linear modeling analyzing MatOb compared to control for each sex separately, controlling for batch effect based on date of harvest. Genes were considered significantly differentially expressed if padj<0.05. NES=Normalized enrichment score; padj = p-value adjusted for multiple testing. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

Unbiased gene set analyses were performed to examine global gene programs altered by exposure to maternal obesity. Overrepresentation analysis revealed that genes with decreased expression in male MatOb offspring compared to male Con are enriched for genes associated with erythrocyte development (Figure 3A). Genes decreased in female MatOb offspring compared to female Con are enriched for programs associated with apoptosis and immunoglobulin production (Figure 3B). Further, immune system and metabolic process programs were significantly enriched in genes that are reduced in MatOb offspring compared to Con regardless of sex (Figure 3A, B). RNA processing and response to stimuli gene programs were overrepresented in genes enriched in female MatOb offspring (Figure 3C), while too few genes were significantly enriched in male MatOb offspring for any gene programs to be overrepresented. These data show common gene programs that were regulated by exposure to maternal obesity regardless of sex as well as striking sex-dependent differences.Figure 3 Treemaps generated using Revigo algorithm showing reduced redundancy gene set analysis for gene ontologies that are significantly enriched in control males compared to MatOB males (A), control females compared to MatOb females (B) and MatOb females compared to control females (C). No gene ontologies were significantly enriched in male MatOb compared to male control. (D) Summary of selected fast gene set enrichment analyses (FGSEA) in male and female pups showing gene signatures in MatOb compared to control that are enriched in previously annotated gene sets (MSigDB). Positive normalized enrichment score indicates the gene set is overrepresented in MatOb compared to control and vice versa. Gene sets are grouped by common annotations. (E–F) Representative FGSEA plots showing changes in interferon responses (E) and interleukin 10 production (F) in MatOb pups compared to Control pups for both male and females. padj = p-value adjusted for multiple testing; N = number of genes from RNA-seq data set that are also in annotated gene set.

Figure 3

To further explore gene programs that were different between male and female MatOb offspring, we performed fast gene set enrichment analysis (fgsea). These analyses revealed that both male and female MatOb offspring had decreased expression of genes associated with metabolic processes and mitochondrial gene programs (Figure 3D). Male MatOb offspring also show strong decrease of cell cycle related genes compared to male Con and an increased expression of cellular development programs, with fewer of these gene sets affected by maternal obesity in female offspring (Figure 3D). Interestingly, one of the most striking findings from the transcriptomic analysis was a marked enrichment in gene programs associated with immune responses, interferon signaling, and cytokine production in female MatOb offspring compared to sex-matched Con that is not seen in male MatOb offspring (Figure 3D). Indeed, there was a strong upregulation of genes associated with IFN-γ signaling and production of cytokines such as IL-10 in female MatOb offspring LSK cells compared to same-sex Con (Figure 3E, F). Male MatOb offspring do not exhibit significant changes and trend toward the opposite effect with respect to these programs compared to same-sex Con (Figure 3E, F). Overall, the RNA-seq revealed sex-differences in differentially expressed genes and enriched gene programs that suggests male MatOb offspring may be more susceptible to develop HSPC dysfunction, and there may be an increase in IFN-γ and/or IL-10 in female MatOb offspring.

3.3 Sex-specific decrease in HSPC number persisted in 8-week offspring

At 8 weeks of age, LT-HSCs remained significantly decreased in male MatOb offspring (Figure 4A) while ST-HSC (Figure 4B) and GMP numbers recovered (Supplemental Fig. 1C). Other progenitors also remained unchanged between groups (Supplemental Fig. 1 A-B, D). Similarly, mature myeloid and lymphoid (CD3 and B220) subsets showed no significant changes in male and female MatOb offspring compared to same-sex Con (Supplemental Fig. 1 E-G). The persistent decrease in LT-HSCs only in male offspring of obese mice at 8 weeks, despite weaning to regular chow, demonstrates sex-specific and persistent impact of maternal obesity exposure on offspring HSPC.Figure 4 Evaluation of offspring at 8 weeks of age. 8-week-old Offspring of obese dams had (A) decreased LT-HSC in a sex-specific manner and (B) unchanged ST-HSC (n = 6–8 mice from 3–4 litters/group).

Figure 4

3.4 Competitive transplantation demonstrates sex-specific differences in engraftment of MatOb offspring HSPCs

Immunophenotypic differences and altered gene programs are not always indicative of functional changes to HSPCs. To further define the functional competency of MatOb offspring HSPCs, we performed a competitive transplantation assay by transplanting sorted Lineage-SCA1+cKIT+ (LSK) BM cells from donor P21 control or MatOb offspring with age- and sex-matched whole BM competitor cells to lethally irradiated mice and assessed early and late engraftment by examining peripheral blood (PB) donor chimerism (Figure 5A). There was no difference in donor chimerism at 4 and 16 weeks after primary transplantation in recipients of either male or female MatOb HSPC when compared to same-sex control (Figure 5B, C). The donor chimerism within BM compartments at 16 weeks post primary transplant was not different (Figure 5D, E).Figure 5 (A) Competitive repopulation model. LSK-enriched BM cells were isolated from CD45.2 Con and MatOb offspring as well as age- and sex-matched CD45.1 competitors. Lethally irradiated CD45.1/CD45.2 recipient mice were injected i. v. with 1000 donor LSK cells and 200,000 BM cells from competitor. PB chimerism was assessed at 4 and 16 weeks. The percentages of donor cells (CD45.2) in PB were determined (B) at 4 weeks and (C) 16 weeks. (D) Chimerism of donor cells within LT-HSC of BM did not show any changes. Data represent the mean ± SEM for n = 14–18 recipient mice collected over three cohorts. (E) 1.0 × 10^6 BM cells isolated from primary recipient mice were transplanted into irradiated F1 mice, PB chimerism was assessed 4- and 16- week post-transplant, and BM was collected at 16 weeks to measure chimerism within LT-HSC. (F–H) PB and BM chimerism after secondary transplantation (n = 10–14/group collected over three cohorts). PB Chimerism of cells originating from male MatOb offspring was significantly lower when comparing to cells from sex- and age-matched male Con at (F) 4- and (G) 16-weeks after secondary transplantation. (H) No significant change was observed in chimerism of all BM cells at 16 weeks, but (I) chimerism of donor cells from male MatOb offspring within BM LT-HSC was significantly decreased, while chimerism of donor cells from female MatOb offspring within BM LT-HSC was increased. 1°, primary; 2°, secondary.

Figure 5

To further stress and assess the long-term function of HSPCs following maternal obesity exposure, we performed a secondary transplantation by transplanting whole BM cells from primary transplantation recipients into lethally irradiated secondary recipient mice 16 weeks after the primary transplantation (Figure 5A). Interestingly, male MatOb offspring HSPCs had significantly decreased secondary engraftment whereas female MatOb offspring HSPC engraftment remained unchanged 4- and 16-weeks after the secondary transplantation (Figure 5F, G). We further assessed chimerism of bone marrow and found that chimerism of all BM cells were unchanged (Figure 5H), but the chimeric proportion of donor LT-HSCs within the BM was significantly decreased in recipients of male MatOb offspring and increased in recipients of female MatOb offspring (Figure 5I). The data indicate that maternal obesity induces sex-specific functional changes in LT-HSC of offspring.

3.5 Increased IL-10 expression in P21 female MatOb offspring recapitulates RNA-seq findings

Given that the enriched gene sets in HSPCs indicated increased cytokine signaling (IFN signaling) and IL-10 production, we next assessed circulating cytokine levels in P21 MatOb offspring. Compared to same-sex controls, at P21, we found that serum IFNα was not different (Figure 6A), but IFNγ was significantly increased in male MatOb offspring (Figure 6B). IL-10 was the only measured cytokine that was significantly increased in P21 female MatOb offspring (Figure 6C). We further assessed serum IL-10 level in primary recipient animals at 16-weeks post-transplant. Similar to P21 offspring, recipient animals received LSK from female MatOb offspring had higher serum IL-10 compared to same-sex Con (Figure 6D). Together these results indicate that exposure to maternal obesity in P21 offspring results in sex-differences in IL-10 and IFNγ levels in offspring.Figure 6 (A–C) Circulating cytokine levels in P21 offspring. (A) No differences in IFN-α expression detected. (B) IFN-ϒ expression was found to be significantly higher in male MatOb offspring compared to same-sex Con while there was no significant change in female offspring. (C) IL-10 expression was increased in female MatOb offspring and unchanged in male. (n = 8–11 from 4 to 5 separate litters/sex/group, ∗p<0.05, ∗∗p<0.01) (D) IL-10 serum in 1° recipient animals showing higher serum IL-10 in recipients of LSK from P21 MatOb female donors. (n = 8–10/sex/group, ∗p<0.05).

Figure 6

3.6 Male MatOb offspring HSPC recipients demonstrated sex-specific changes in metabolic phenotype

We next assessed the metabolic phenotype of recipient animals to determine the role of HSPCs in regulating total body glucose tolerance. Sixteen weeks after the primary transplant, no difference in body weight or adiposity of recipient mice was observed (Figure 7A, B). Surprisingly, the recipient mice exhibited sex-differences in glucose intolerance. Specifically, recipients of male MatOb HSPC developed glucose intolerance (Figure 7C, E), with 15.6 ± 4.7 % increase in area under the curve (AUC) of glucose tolerance test (GTT) compared to same-sex controls that received HSPCs from Con pups. In contrast, female recipients of MatOb HSPC recipients were not different compared to same-sex controls (Figure 7D, E). Insulin tolerance tests (ITT) showed no difference (Figure 7F, G), indicating that insulin resistance may not be the primary contributor of glucose intolerance. Therefore, we further assessed beta cell health by assessing pancreatic beta cell area (Figure 7H,I). Compared to recipients of same-sex Con, there was a trend towards lower pancreatic beta cell area in male MatOb HSPC recipients (Figure 7H, p = 0.0562) and higher pancreatic beta cell area in female MatOb HSPC recipients (Figure 7I). These findings indicate the role of HSPCs in regulating glucose metabolism potentially by influencing beta cell health in primary recipient mice.Figure 7 Primary transplant recipient mice with LSK cells from Con and MatOb offspring had similar (A) body weight and (B) body adiposity. (n = 7–8/sex/group) (C) Recipients of male MatOb offspring LSK cells had significant increase in glucose excursion curve during GTT (n = 13/group), while (D) recipients of female MatOb offspring LSK cells did not show any difference in GTT comparing with recipients of female Con (n = 16–18/group). Area under curve of GTT AUC (E). (F–G) MatOb offspring LSK recipients did not show any difference in changes to ITT compared to Con (n = 6–10/sex/group). GTT: glucose tolerance test; ITT: insulin tolerance test. (H–I) Pancreatic beta cell area of primary recipients. (H) Male MatOb offspring LSK recipients had lower pancreatic beta cell area (p = 0.047 by unpaired t-test, n = 13–15 animals/group), while (I) female MatOb offspring LSK recipients had an increase in beta cell area (p = 0.045 by unpaired t-test, n = 12–13/group). ∗p<0.05.

Figure 7

Following the secondary transplantation, body weight and adiposity of the recipient mice again remained unchanged (Figure 8A, B). Interestingly, not only did we observe the similar pattern of sex-differences in glucose intolerance, the extent of glucose intolerance in secondary transplant recipient mice of male MatOb offspring HSPCs was even more prominent (27.7 ± 8.9% increase in GTT AUC vs. same-sex controls) (Figure 8C). In contrast, recipients of female MatOb HSPCs did not have any significant differences in GTT compared to the control group (Figure 8D). Similar to the primary transplant, MatOb offspring recipients did not have any difference in changes to insulin challenge (Figure 8E, F).Figure 8 Con and MatOb secondary transplant recipient mice had unchanged (A) body weight and (B) body adiposity. (C) Secondary transplant recipients of male MatOb offspring had worsening GTT (n = 9/group) whereas (D) secondary transplant recipients of female MatOb offspring GTT was not significantly different than same-sex Con (n = 13–14/group). (E–F) No difference in ITT of secondary transplant recipients regardless of donors (n = 3–4/sex/group). ∗p<0.05.

Figure 8

4 Discussion

This study illustrates that maternal obesity induces changes in the characteristics of hematopoietic cells in offspring as early as postnatal day 21. Numerous studies have presented evidence of the changes in the landscape of fetal immune cells in response to maternal obesity exposure. In humans, cord blood mononuclear cells from infants born to mothers with obesity were found to exhibit increased CD4+ T-cells and reduced myeloid cell population [27]. Epigenomic changes favoring effector memory cells were observed in CD4+ T-cells, along with reduced cytokine production in response to stimulation [26]. Another cohort study reported increased lymphocyte subsets in umbilical cord blood from mothers with obesity, while CD34+ cells, which contain HSPCs, were decreased [30]. Additionally, fetal monocyte and dendritic cell responses to inflammatory stimuli were reduced in pregnancies complicated by obesity [23]. Collectively, these findings relay the possibility of altered long-term HSPC function in offspring exposed to maternal obesity. Studies conducted on mice investigating the acute effects of a high-fat or Western diet during pregnancy demonstrate compromised self-renewal and myeloid-biased differentiation in fetal liver HSPCs [19]. Similarly, exposure to a maternal Western diet induces fetal HSPC reprogramming through bone marrow adipogenesis in rhesus macaques [20]. Another study on nonhuman primates revealed that in-utero exposure to a maternal Western diet promoted epigenetic reprogramming of HSPCs towards a pro-inflammatory phenotype that persists up to 3 years of age, even after transitioning to a regular diet [12]. We found that while multiple cell lineages were affected during early life, the only persistent change observed at 8 weeks of age was decreased LT-HSCs in male animal offspring exposed to maternal obesity. Additionally, through competitive transplant experiments, we observed altered long-term function of HSPCs in a sex-specific manner. These findings align with recent reports indicating that maternal obesity leads to HSPC dysfunction, and further highlights the importance of assessing sex independently. Importantly, our results showing impaired HSPC after transplanting to non-obese recipients that that hematopoietic dysfunction may originate from early changes in HSPCs due to maternal obesity exposure, rather than being a consequence of offspring adiposity. This observation is also relevant in the context of DOHaD when considering the evolutionary mismatch hypothesis, which proposes that a mismatch between evolutionary history and current environment may contribute to disease risks particularly during critical developmental windows [31,32]. In our study, the offspring were exposed to altered in-utero and postnatal environments due to maternal obesity, while the offspring and recipient animals were fed regular chow. This discrepancy between maternal diet and postnatal diet could potentially influence the observed HSPC dysfunction and metabolic effects. Therefore, a valuable future research direction would be to explore how aligning maternal and offspring diets might impact HSPC function and metabolic health.

Transcriptomic analyses conducted of LSK cells from postnatal day 21 offspring revealed intriguing observations regarding the differential gene expression between female and male offspring. It was observed that 72% of the genes exhibiting differential expression in male pups were also differentially expressed in females. However, in addition to this overlap, several genes displayed significant differential expression exclusively in female pups. One of the remarkable discoveries from the transcriptomic analysis was the noticeable enrichment of gene programs associated with immune responses, interferon signaling, and cytokine production specifically in female MatOb offspring compared to sex-matched controls, while such differences were absent in male MatOb offspring. Particularly, there was a strong increase in expression of genes linked to IFNγ signaling and the production of cytokines like IL-10 in female MatOb offspring compared to the control group. Male MatOb offspring did not exhibit significant changes and even displayed the opposite trend in these programs in comparison to the control group. This finding suggests that although maternal obesity affects the hematopoietic transcriptome of offspring regardless of sex, the short-term production of specific anti-inflammatory signaling molecules, such as IL-10, may exert a protective effect against HSPC dysfunction in female MatOb offspring. This observation, in turn, may contribute to sex-dependent differences in the long-term effects of maternal diet on hematopoietic cells. In this study, we report, for the first time, sex-specific alterations in offspring IL-10 production due to maternal obesity exposure and the increase in IL-10 was recapitulated in the primary recipient that received LSK from female MatOb offspring.

Animal studies indicate maternal obesity exposure has sex-specific effects on offspring health [13,33,34]. Despite this known sex-specific impact, the molecular mechanisms underlying this sex bias remain largely unknown, but the sex bias in impaired pancreatic β-cell function in adults can be attributed to lower tolerance to endoplasmic reticulum stress in male islets and the protective effects of estrogen in females [[35], [36], [37]]. In contrast, sex-differences observed in neuropsychiatric diseases in response to maternal exposure is influenced by a complex interplay of various contributing factors [38,39]. One theory suggests the placenta of male fetuses is more reactive to maternal stress, while another proposes decreased expression of X-linked methylation genes in males leads to a more responsive chromatin state [34,40,41]. Nonetheless, our findings support the vulnerability of male offspring to effects of maternal obesity exposure, potentially due to the lack of protective pathways present in female offspring.

Apart from highlighting the sex-specific alterations in offspring HSPC function resulting from maternal obesity, our findings revealed a novel role of HSPCs in the regulation of total body glucose tolerance. Our previous study demonstrated that the offspring of obese dams exhibited sex-specific differences in glucose intolerance and islet insulin secretion [13]. Specifically, male offspring developed glucose intolerance and islet dysfunction. In this study, male MatOb LSK cell primary and secondary transplantation recipients developed glucose intolerance, while recipients of female MatOb LSK cells did not. In humans, HSPCs may play a crucial role in type 1 diabetes (T1DM) and type 2 diabetes (T2DM), both of which are diseases related to impaired pancreatic beta cell function resulting in glucose intolerance and hyperglycemia. Two recent metanalyses reported that patients with T1DM and T2DM had improvement in fasting blood glucose and a higher C-peptide following HSPC transplantation, indicating improvement in disease state due to recovery of beta cell function [42,43]. Of note, BM HSPCs were found to have better efficacy in reducing fasting blood glucose than the anti-diabetic drug semaglutide [43]. Recently, Chang et al. demonstrated that co-transplanting islets with donor HSPC reversed hyperglycemia in a preclinical model of diabetes [44]. Consistent with these findings, we found that recipients transplanted with HSPCs from male MatOb pups had a trend towards a lower pancreatic beta cell area, suggesting that glucose intolerance in recipients of male MatOb pups HSPC is secondary to impaired beta cell function. Future studies will focus to perform in-depth assessment of beta cell islet insulin secretion using advanced methods such as islet perifusion, as well as to delineate the mechanisms contributing to lower beta cell area. These investigations will further elucidate the role of HSPCs in glucose metabolism and their potential therapeutic implications for diabetes development.

Our data collectively unveil a sex-specific alteration in HSPC function among offspring of obese mice, consistent with the observed pattern of glucose intolerance in recipients of LSK-enriched cells. Moreover, this study demonstrates the evidence supporting HSPCs’ involvement in regulating glucose metabolism by modulating beta cell health. Our future experiments will delve into the mechanisms contributing to higher IL-10 in female offspring of obese mice, and the direct role of IL-10, in both aspect of HSPC dysfunction and glucose intolerance in offspring of obese mice.

5 Conclusion

In conclusion, maternal obesity induces sex-specific differences in transcriptomes of offspring HSPC and leads to sex-differences in HSPC function. HSPC dysfunction may be one of the mechanisms contributing to metabolic dysfunction seen in offspring exposed to maternal obesity.

Data sharing statement

For original data, please contact Kok Lim Kua, MD (kkua@iu.edu) and Maegan Capitano, PhD (malcapit@iu.edu). RNA sequencing raw and processed data are available on Gene Expression Omnibus (GEO, GSE253870.

CRediT authorship contribution statement

Merve Denizli: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. James Ropa: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Lindsay Beasley: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Joydeep Ghosh: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Kelli DeVanna: Methodology, Investigation, Data curation. Taylor Spice: Methodology, Investigation, Data curation. Laura S. Haneline: Writing – review & editing, Investigation, Conceptualization. Maegan Capitano: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Kok Lim Kua: Writing – review & editing, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kok Lim Kua, Maegan capitano reports financial support was provided by 10.13039/100000002 National Institutes of Health . Kok Lim Kua reports financial support was provided by 10.13039/100000912 March of Dimes Foundation . Kok Lim Kua reports financial support was provided by Riley Children's Foundation. James Ropa reports financial support was provided by 10.13039/100000002 National Institutes of Health . Laura Haneline reports financial support was provided by Riley Children's Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Supplemental Table 1 and Table 2

Diffenretially Expressed Genes comparing MatOb Male and Female Offspring to same-sex controls

Supplemental Table 1 and Table 2

Supplemental Methods

Supplemental Methods

Supplemental Figure 1

Supplemental Figure 1

Data availability

Data will be made available on request.

Acknowledgement

The graphical abstract was made using BioRender.com. We thank the Translational Core of the Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine for assistance in performing the cytokine analysis using MSD platform, and the Indiana University Center for Medical Genomics for performing RNA-sequencing. MC was funded by 10.13039/100000002 NIH 10.13039/100000062 NIDDK (U54DK106846 ). KLK was funded by Showalter Research Trust Fund, Riley Children's Foundation, Pilot and Feasibility Award within the 10.13039/100008574 CDMD 10.13039/100000002 NIH 10.13039/100000062 NIDDK Grant Number (P30DK097512 ), 10.13039/100000912 March of Dimes Foundation Basil O'Connor Award and 10.13039/100000002 NIH NICHD (K08HD109636 ). JR was funded by 10.13039/100000002 NIH 10.13039/100000050 NHLBI (F32HL160072 , K99HL166790 ). 10.13039/100016036 LSH was funded by Riley Children's Foundation. Support for article processing charges provided by the IU Open Access Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.molmet.2024.102008.
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