The GLP-1 analog, liraglutide prevents the increase of proinflammatory mediators in the hippocampus of male rat pups submitted to maternal perinatal food restriction

Abstract

Background: Perinatal maternal malnutrition is related to altered growth of tissues and organs. The nervous system development is very sensitive to environmental insults, being the hippocampus a vulnerable structure, in which altered number of neurons and granular cells has been observed. Moreover, glial cells are also affected, and increased expression of proinflammatory mediators has been observed. We studied the effect of Glucagon-like peptide-1 receptor (GLP-1R) agonists, liraglutide, which have very potent metabolic and neuroprotective effects, in order to ameliorate/prevent the glial alterations present in the hippocampus of the pups from mothers with food restriction during pregnancy and lactation (maternal perinatal food restriction-MPFR).

Methods: Pregnant Sprague-Dawley rats were randomly assigned to 50% food restriction (FR; n = 12) or ad libitum controls (CT, n = 12) groups at day of pregnancy 12 (GD12). From GD14 to parturition, pregnant FR and CT rats were treated with liraglutide (100 μg/kg) or vehicle. At postnatal day 21 and before weaning, 48 males and 45 females (CT and MPFR) were sacrificed. mRNA expression levels of interleukin-1β (IL1β), interleukin-6 (IL-6), nuclear factor-κβ, major histocompatibility complex-II (MHCII), interleukin 10 (IL10), arginase 1 (Arg1), and transforming growth factor (TGFβ) were assessed in the hippocampus by quantitative real-time polymerase chain reaction. Iba1 and GFAP-immunoreactivity were assessed by immunocytochemistry.

Results: The mRNA expression IL1β, IL6, NF-κB, and MHCII increased in the hippocampus of male but not in female pups from MPFR. In addition, there was an increase in the percentage of GFAP and Iba1-immupositive cells in the dentate gyrus compared to controls, indicating an inflammatory response in the brain. On the other hand, liraglutide treatment prevented the neuroinflammatory process, promoting the production of anti-inflammatory molecules such as IL10, TGFβ, and arginase 1, and decreasing the number and reactivity of microglial cells and astrocytes in the hippocampus of male pups.

Conclusion: Therefore, the GLP-1 analog, liraglutide, emerges as neuroprotective drug that minimizes the harmful effects of maternal food restriction, decreasing neuroinflammation in the hippocampus in a very early stage.

Keywords: Astroglia; Cytokines; GLP-1; Hippocampus; Incretins; Inflammation; Liraglutide; Maternal food restriction; Microglia.

Fig. 1

Body weight gain of pregnant dams throughout the food restriction protocol, from mating until day 21 of gestation. Pregnant rats were randomly submitted to food restriction (50% of daily intake of control dams/MPFR) from gestational day 12 or were fed ad libitum in the control group (CT; see arrow). From gestational day 14 to parturition, dams were treated with liraglutide (LIRA; 100 μg/kg/12 h, see arrow) or vehicle. Data are mean + SEM. (MPFR/VEH compared to CT/VEH: *p < 0.05, **p < 0.01, ****p < 0.0001; MPFR/VEH compared to CT/LIRA: ^&p < 0.05, ^&&p < 0.01, ^&&&&p < 0.0001; MPFR/LIRA compared to CT/VEH: ^aap < 0.01, ^aaap < 0.001, ^aaaap < 0.0001; MPFR/LIRA compared to CT/LIRA: ^bbbp < 0.001, ^bbbbp < 0.0001)

Fig. 2

a Body weight of male pups at PD21. b Body weight of female pups at PD21. CT/VEH, control pups treated with vehicle; CT/LIRA, control pups treated with liraglutide; MPFR/VEH, food-restricted pups treated with vehicle; MPFR/LIRA, food-restricted pups treated with liraglutide. Data are mean + SEM. (*p < 0.05 compared to CT/VEH; ***p < 0.001 compared to CT/VEH; ****p < 0.0001 compared to CT/VEH; ^&p < 0.05 compared to CT/LIRA; ^&&p < 0.01 compared to CT/LIRA; ^&&&p < 0.001 compared to CT/LIRA)

Fig. 3

a Representative images of Iba-immunoreactivity in the dentate gyrus of male pups. Scale bar, 20 μm. b Quantification of Iba-1 immunoreactivity in the dentate gyrus in male pups. c Representative images of Iba-immunoreactivity in the dentate gyrus of female pups. Scale bar, 45 μm. d Quantification of Iba-1 immunoreactivity in the dentate gyrus in female pups. CT/VEH, control pups treated with vehicle; CT/LIRA, control pups treated with liraglutide; MPFR/VEH, food-restricted pups treated with vehicle; MPFR/LIRA, food-restricted pups treated with liraglutide. Data are mean + SEM. (**p < 0.01 compared to CT/VEH; ***p < 0.001 compared to CT/VEH; ^&&p < 0.01 compared to CT/LIRA; ^&&&&p < 0.0001 compared to CT/LIRA; ^#p < 0.05 compared to MPFR/VEH; ^##p < 0.01 compared to MPFR/VEH)

Fig. 4

Morphological changes of Iba-1 immunoreactive cells in the hilus of dentate gyrus of the hippocampus. a The upper panel show examples of the morphological types in which cells were classified. Type I, cells with few cellular processes; type II, cells showing four short branches; type III, cells with numerous cell processes and a small cell body; type IV, cells with large somas and retracted and thicker processes; and type V, cells with numerous short processes and intense Iba1 immunostaining. b The graph shows the proportion of reactive cells (types IV and V) in CT/VEH, control pups treated with vehicle; CT/LIRA, control pups treated with liraglutide; MPFR/VEH, food-restricted pups treated with vehicle; and MPFR/LIRA, food-restricted pups treated with liraglutide. Data are mean + SEM. (***p < 0.001 compared to CT/VEH; ^&&p < 0.01 compared to CT/LIRA; ^##p < 0.01 compared to MPFR/VEH)

Fig. 5

a Representative images of GFAP-immunoreactivity in the dentate gyrus of male pups. Scale bar, 30 μm. b Quantification of GFAP-1 immunoreactivity in the dentate gyrus in male pups. c Representative images of GFAP-immunoreactivity in the dentate gyrus of female pups. Scale bar, 45 μm. d Quantification of GFAP-1 immunoreactivity in the dentate gyrus in female pups. CT/VEH, control pups treated with vehicle; CT/LIRA, control pups treated with liraglutide; MPFR/VEH, food-restricted pups treated with vehicle; and MPFR/LIRA, food-restricted pups treated with liraglutide. Data are mean + SEM. (*p < 0.05 compared to CT/VEH; ^&p < 0.05 compared to CT/LIRA; ^#p < 0.05 compared to MPFR/VEH)

Fig. 6

Proinflammatory mediators in male offspring. a Interleukin-1b (IL1b). b Interleukin-6 (IL-6). c Nuclear factor-κB (NF-κB). d Major histocompatibility complex-II (MHCII). e Interleukin-10 (IL10). f Transforming growth factor β (TGFβ). g Arginase 1 (ARG1) mRNA levels in the hippocampus. CT/VEH, control pups treated with vehicle; CT/LIRA, control pups treated with liraglutide; MPFR/VEH, food-restricted pups treated with vehicle; MPFR/LIRA, food-restricted pups treated with liraglutide. Data are mean + SEM. (*p < 0.05 compared to CT/VEH; **p < 0.01 compared to CT/VEH; ^&p < 0.05 compared to CT/LIRA; ^&&p < 0.01 compared to CT/LIRA; ^#p < 0.05 compared to MPFR/VEH; ^##p < 0.01 compared to MPFR/VEH)

Fig. 7

Proinflammatory mediators in female offspring. a Interleukin-1b (IL1b). b Interleukin-6 (IL-6). c Nuclear factor-κB (NF-κB). d Major histocompatibility complex-II (MHCII). e Interleukin-10 (IL10). f Transforming growth factor β (TGFβ). g Arginase 1 (ARG1) mRNA levels in the hippocampus. CT/VEH, control pups treated with vehicle; CT/LIRA, control pups treated with liraglutide; MPFR/VEH, food-restricted pups treated with vehicle; MPFR/LIRA, food-restricted pups treated with liraglutide. Data are mean + SEM. (^&p < 0.05 compared to CT/LIRA; ^#p < 0.05 compared to MPFR/VEH)

Fig. 8

Increased number of Iba-1-immunopositive cells (microglia) was observed in the maternal perinatal food restriction (MPFR) model. Increased mRNA expression of proinflammatory factors: major histocompatibility complex II (MHC type II) molecules, Interleukin-1β (IL-1β), interleukin-6 (IL-6), and the transcription factor NF-κB. The administration of liraglutide to malnourished pregnant rats promoted a protective microglial phenotype in the litters, decreasing the reactivity and number of Iba-1-immunopositive cells (microglia), able of producing protective and anti-inflammatory factors: interleukin-10 (IL-10), transforming growth factor-β (TGF-β) or arginase 1 (Arg1). These effects were just observed in males