Impact of selected breast milk lipid species and miRNAs on insulin sensitivity programming in a rodent model of gestational diabetes

Abstract :

According to the concept of Developmental Origins of Health and Disease (DOHaD), a deleterious perinatal environment may condition the later onset of metabolic diseases. Gestational Diabetes Mellitus (GDM) represents a growing public health concern with lasting consequences in offspring, as insulin resistance (IR)-related metabolic disorders. Breastfeeding (BF) is increasingly recognized as a critical window for mitigating the long-term ‘programming effect’ of GDM. These benefits are likely mediated by molecular determinants of breast milk (BM) susceptible to counteract the gluco- and lipo-toxicity effects of intrauterine hyperglycemia on fetal β-cells and liver.

In an ongoing project funded by French research agency (ANR-22-CE17-0039-GDM-MILK), we explore the complex interplay between maternal hyperglycemia during gestation, GDM-related BM composition, and its long-term sex-specific effects on developmental and metabolic outcomes in offspring using a cross-fostering rodent GDM-model. In this model, maternal impaired glucose tolerance (IGT) during gestation was associated with a unique BM- composition, related to GDM and its evolution following delivery, with enhanced content in choline-related lipid species and miRNAs, reported to present emergent roles in insulin signaling.

Adult male and female offspring born to GDM-dams but nursed with milk from control dams presented reduced insulin secretion and sensitivity, respectively, in response to a hypercaloric challenge, an effect not observed in those nursed by GDM-dams.

The overall objective of the PhD project is to decipher the contribution of specific GDM-BM bioactive lipid species and miRNAs, in the modulation of energy homeostasis and insulin signaling pathway in male and female offspring exposed to hyperglycemia in utero (‘first hit’), throughout their life.

The first objective is to evaluate the effects of these molecules in vitro using pancreatic and enteroendocrine cell lines, in order to elucidate their roles in the regulation of energy homeostasis.

The second objective is to assess the in vivo effects of these candidate molecules in male and female offspring from our pre-clinical GDM-rodent model, by evaluating their metabolic responses to a ‘second IR-related hit’ (either a hypercaloric diet (males and females) or pregnancy in cases of females).

Enjeux du projet :

1. Contexte et enjeux scientifiques

According to the concept of Developmental Origins of Health and Disease (DOHaD), a deleterious perinatal environment may condition the later onset of metabolic diseases. Gestational Diabetes Mellitus (GDM) represents a growing public health concern, affecting 16.7% of pregnancies worldwide, with lasting consequences in offspring, and this, despite maternal ante-natal interventions. Indeed, GDM may contribute to an intergenerational cycle of insulin resistance (IR)-related metabolic disorders such as type 2 diabetes (T2D) and Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). Breastfeeding (BF) is increasingly recognized as a critical window for mitigating the long-term ‘programming effect’ of GDM, with a 18% reduction in prediabetes-risk among breastfed infants born to GDM-women. These benefits are likely mediated by molecular determinants susceptible to counteract the gluco- and lipo-toxicity effects of intrauterine hyperglycemia on fetal β-cells and liver. Despite this potential, fewer than 30% of infants born to GDM-mothers are breastfed beyond 12 months, underscoring the urgent need to provide robust mechanistic evidence supporting the benefits of BF and to implement targeted interventions that promote sustained BF in this high-risk population.

In an ongoing project funded by French research agency (ANR-22-CE17-0039-GDM-MILK), we explore the complex interplay between maternal hyperglycemia during gestation, and its associated metabolic status - GDM-related BM composition, as well as its long-term sex-specific effects on developmental and metabolic outcomes in offspring using a cross-fostering rodent GDM-model. In this model, animals showed maternal impaired glucose tolerance (IGT) during gestation, maintained or not during lactation, associated with β-cells hyperplasia in newborns. Maternal IGT resolution postpartum was associated with an unique breast milk composition, related to GDM and its evolution following delivery, with enhanced content in choline-related lipid species and miRNAs, reported to present emergent roles in insulin signaling. Adult male and female offspring born to GDM-dams but nursed with milk from control dams presented reduced insulin secretion and sensitivity, respectively, in response to a hypercaloric challenge, an effect not observed in those nursed by GDM-dams.

Based on these findings, we hypothesize these specific bioactive components (selected lipid species and miRNAs) of GDM-related BM could influence insulin signaling in offspring exposed in utero to hyperglycemia (‘first hit’), in a sex-dependent manner. To test this hypothesis, we will administer a daily supplementation of these bioactive compounds, from birth to weaning, to offspring exposed in utero to hyperglycemia but receiving non-adapted milk, as they will be cross-fostered and nursed by control dams. As previously demonstrated but in the absence of postnatal supplementation, these offspring exhibit reduced insulin sensitivity in adulthood. To evaluate the putative effects of these BM bioactive compounds as regulators of metabolic homeostasis, we will longitudinally monitor the metabolic trajectory of the supplemented pups from early life, through adolescence, and into adulthood. Into adulthood, rats will be challenged with a ‘second hit known to promote IR-onset’ (either a hypercaloric diet (males and females) or pregnancy in cases of females). This approach will allow us to assess offspring metabolic flexibility as well as the intergenerational risk of T2D or GDM, in response to offspring postnatal supplementation of the BM candidates.

Hormone secretion in response to these lipid species and miRNAs administration, as well as the molecular mechanisms that contribute to β-cell and enteroendocrine cell (EEC) plasticity, will be assessed in isolated islets of Langerhans from offspring of our GDM-model and in vitro in insulin and incretin-producing cell lines. In vivo, an integrated physiological approach will be used to study glucose homeostasis, insulin sensitivity, β-cell mass dynamics, pancreatic plasticity and EEC function in pups exposed to GDM in utero and postnatally supplemented in adulthood in response to the ‘second IR-related hit’.

Overall, this project aims to pave the way to optimize breastfeeding strategies and maternal nutritional interventions in GDM-high risk population, thereby offering a potential strategy to limit the burden of metabolic disease (T2D, MASDL), in future generations.

2. Mots‑clés : Gestational Diabetes Mellitus, DOHaD, offspring metabolic programming, lactation, milk bioactive compounds, insulin signalling, MASLD, type 2 diabetes.

3. Objectifs du projet

The overall objective of the PhD project is to decipher the contribution of specific GDM-BM bioactive lipid species and miRNAs, in the modulation of energy homeostasis and insulin signaling pathway in offspring exposed to hyperglycemia in utero, throughout their life.

The first objective is to evaluate the effects of these molecules in vitro using pancreatic and enteroendocrine (EEC) cell lines, in order to elucidate their roles in the regulation of energy homeostasis.

The second objective is to assess the in vivo effects of these candidate molecules in male and female offspring from our pre-clinical GDM-rodent model, by evaluating their metabolic responses to a ‘second IR-related hit’.

Approches et Méthodologie et plan de travail

To address these questions, we have implemented a validated pre-clinical nutritional GDM-rodent model with maternal impaired glucose tolerance (IGT) resolved or not postpartum (Bobin et al, 2025) and with cross-fostering, not ethically transposable in humans. This GDM-model allows to analyze independently the impact of both pre- and post-natal periods on the metabolic outcome of offspring, in relation to the BM they received in early life.

Part A. In vitro investigation of β-cell and incretin-producing cell lines plasticity in response to breast milk-derived bioactive lipid and miRNAs (Year 1).  

To address the first objective of the project, establish pancreatic β-cell and EEC lines will be supplemented in vitro with the candidate molecules. Functional assays will be conducted to assess insulin secretion, glucose responsiveness, and cell viability. In parallel, the underlying molecular mechanisms will be investigated, including oxidative stress, mitochondrial dysfunction, and inflammatory signaling pathways, that may contribute to β-cell failure and its putative correction.

In EEC models, cellular differentiation, secretory function and gene expression profiles will be evaluated to determine their contribution to glucose regulation in the gut–pancreas communication.

Together, these complementary approaches will provide mechanistic insights into the role of breast-milk derived bioactive compounds to modulate key cellular processes involved in metabolic regulation.

Part B.  Postnatal in vivo supplementation of breast milk-derived lipid and miRNAs: impact on metabolic flexibility and adaptation to a second insulin-resistance challenge (Years 1-2).

The second part of the PhD project aims to determine whether postnatal supplementation with selected breast milk-derived lipid species and miRNAs can beneficially or detrimentally modulate metabolic flexibility in offspring born to GDM mothers. The effects will be evaluated both during early life and in adulthood, particularly in response to a second IR-related challenge.

To address this objective, a longitudinal integrative physiological approach will be implemented in male and female offspring born to GDM dams, cross-fostered by control dams, and orally supplemented from birth to weaning with a defined mixture of candidate BM biomarkers.  Specifically, pancreatic, enteroendocrine, and hepatic metabolic adaptations will be assessed through level measurements of insulin, gastrointestinal hormones (including incretins), adipokines, cytokines, and growth factors using ELISA immunoassays.

In parallel, mature pancreatic β-cells, enteroendocrine, and hepatic functions will be investigated across developmental stages. At the tissue level, mechanistic analyses will combine single-cell transcriptomic profiling with targeted miRNA expression analysis using RT-qPCR, complemented by immunohistochemical approaches. Systemic metabolic trajectories will be characterized using longitudinal metabolomics and lipidomics analysis in blood and tissues, integrated with physiological assessments of insulin sensitivity.

Finally, within this whole-body framework, the role of microbial metabolites will be explored regarding its putative impact on β-cells and EEC activity in the context of GDM-associated metabolic programming.  

Part C. Integrated longitudinal analysis of metabolic trajectories and insulin-sensitivity of GDM-offspring postnatally supplemented in BM-lipid species and miRNAs: data integration and thesis-valorization (Year 3).

A comprehensive multi-sources data integration will be implemented, encompassing various biological matrices, including blood and tissues (pancreas, liver, intestine cells, adipocytes), and multi-omics datasets (metabolomic, lipidomic, gene expression and physiological data). The temporal dimension of the experimental design will be taken account to characterize dynamic metabolic trajectories across the lifespan.

To this end, innovative data analysis methods will be employed, including ANOVA–Simultaneous Component Analysis (ASCA) and related frameworks, as well as Path Modelling approaches.

The results obtained within the PhD project will be valorized through at least two scientific publications with the doctoral candidate as first author, together with communications at international conferences (International Society of DOHaD, International Diabetes,etc).

Perspectives et impact du projet

The expected outcomes of the PhD project are: 1) the in vitro and in vivo validation of specific choline-lipid species and miRNAs identified in the breast milk of GDM mothers, that possess the potential to modulate pancreatic and/or intestinal enteroendocrine cell function and activity, which produce or stimulate insulin release, respectively;  2) the characterization of the effects of post-natal supplementation with these BM-derived candidates on pancreas, EEC, liver, and adipose tissue homeostasis in offspring born to GDM-mothers; and more broadly 3) the evaluation of the offspring‘s metabolic trajectory, with a focus on insulin signaling pathway, while accounting for sex-specific differences. Moreover, these selected BM-lipid species and miRNAs will also be validated in human BM biocollection in an ongoing GDM-cohort (ANR-22-CE17-0039-GDM-MILK).

The project aims to deliver a significant breakthrough by establishing the lactation period as a critical window for sustainable intervention, with a potential to mitigate the growing burden of insulin resistance-related metabolic diseases in future generations, particularly within the vulnerable population of infants born to diabetic mothers. 

Encadrement

• Directrice de thèse : Marie-Cécile Alexandre-Gouabau, PhD, HDR (UMR 1280 PhAN) (expertises : metabolic programming, DOHaD, lactation, breast milk composition ; insulin sensitivity in rodent programming models)

• Co‑encadrantes : Gwenola Le Dréan, PhD, HDR (UMR 1280 PhAN) (expertises : gut-brain axis, DOHaD, intestinal physiology in rodent programming, EECs), et Sandrine Le Guillou, PhD [UMR 1313 Génétique Animale et Biologie Intégrative (GABI), Equipe Etudes Fonctionnelles et Modèles Innovants pour la Santé des Animaux d'Elevage (EFISA)] (expertise in breast milk composition, mammary gland physiology, and miRNAs using high throughput analysis)

Contact :

N° de tél : : 02 53 48 20 12

Mail : Marie-Cecile.Alexandre-Gouabau@univ-nantes.fr

Équipe d’accueil

• Laboratoire d’accueil : UMR 1280 – PhAN (Physiopathologie des Adaptations Nutritionnelles)
• Axe scientifique : Axe A Développement foetale, croissance, devenir métabolique et cognitif

The project is supported by funding obtained from the French National Research Agency (ANR-22-CE17-0039-GDM-MILK). The research unit has access to: 1) a rodent animal facility and skilled technicians for the implementation and monitoring of perinatal malnutrition models; 2) mass spectrometry facility for metabolites (MELISA-LABERCA and M Shark plateforms); 3) two cell culture rooms and molecular biology equipment managed by a research engineer and supervised by Gwenola Le Dréan, who is a co-supervisor of the PhD project. The UMR PhAN also has access to cytology, and sequencing platforms at the UMS Biocore facility at Nantes University.

Sandrine Le Guillou (UMR 1313, GABI), who is a co-supervisor of the PhD project, has access to outsourcing / subcontracting for human and rat milk miRNome libraries and sequencing (outsourcing-GenomEast plateform). These libraries will be used in this project.

Innovative data analysis methods of multi-sources omics integration with temporal dimension factor, have already developed by the partner StatSC of the ANR GDM-MILK consortium.

Master’s degree in pathophysiology with solid knowledge of metabolism and physiology; strong interest in integrative physiology approaches; proficiency in standard laboratory techniques, including cell culture, molecular biology, and Western blotting. Experience in comprehensive molecular profiling (metabolomics, lipidomics). Interest in “omics data” analysis. An interest in both in vitro and in vivo experimentation would be appreciated. Certification in animal experimentation design is required, although training can be provided if needed.