Maternal overnutrition by hypercaloric diets programs hypothalamic mitochondrial fusion and metabolic dysfunction in rat male offspring

Abstract

Background: Maternal overnutrition including pre-pregnancy, pregnancy and lactation promotes a lipotoxic insult leading to metabolic dysfunction in offspring. Diet-induced obesity models (DIO) show that changes in hypothalamic mitochondria fusion and fission dynamics modulate metabolic dysfunction. Using three selective diet formula including a High fat diet (HFD), Cafeteria (CAF) and High Sugar Diet (HSD), we hypothesized that maternal diets exposure program leads to selective changes in hypothalamic mitochondria fusion and fission dynamics in male offspring leading to metabolic dysfunction which is exacerbated by a second exposure after weaning.

Methods: We exposed female Wistar rats to nutritional programming including Chow, HFD, CAF, or HSD for 9 weeks (pre-mating, mating, pregnancy and lactation) or to the same diets to offspring after weaning. We determined body weight, food intake and metabolic parameters in the offspring from 21 to 60 days old. Hypothalamus was dissected at 60 days old to determine mitochondria-ER interaction markers by mRNA expression and western blot and morphology by transmission electron microscopy (TEM). Mitochondrial-ER function was analyzed by confocal microscopy using hypothalamic cell line mHypoA-CLU192.

Results: Maternal programming by HFD and CAF leads to failure in glucose, leptin and insulin sensitivity and fat accumulation. Additionally, HFD and CAF programming promote mitochondrial fusion by increasing the expression of MFN2 and decreasing DRP1, respectively. Further, TEM analysis confirms that CAF exposure after programing leads to an increase in mitochondria fusion and enhanced mitochondrial-ER interaction, which partially correlates with metabolic dysfunction and fat accumulation in the HFD and CAF groups. Finally, we identified that lipotoxic palmitic acid stimulus in hypothalamic cells increases Ca²⁺ overload into mitochondria matrix leading to mitochondrial dysfunction.

Conclusions: We concluded that maternal programming by HFD induces hypothalamic mitochondria fusion, metabolic dysfunction and fat accumulation in male offspring, which is exacerbated by HFD or CAF exposure after weaning, potentially due to mitochondria calcium overflux.

Keywords: Diet induced obesity (DIO); Fission; Fusion; Hypothalamus; Maternal overnutrition; Mitochondria; Mitochondria dynamics.

Fig. 1

Effect of nutritional programming on dams and male offspring weight. a) Animal model. We fed female Wistar rats for 9 weeks according to the schedule to promote maternal programming or we fed offspring with hypercaloric diet exposure after weaning. b) Maternal body weight was followed for 6 weeks (pre-mating, mating and gestation) (Control n = 7, HFD n = 7, CAF n = 6 and HSD n = 4). c) Birth weight of offspring at day 0. d) Body weight after weaning for Control versus HFD-C, CAF-C, HSD-C for maternal programming groups, where HFD, CAF and HSD are maternal diet and C is control chow diet of offspring. e) Body weight after weaning for Control versus offspring fed with HFD, CAF and HSD after weaning (HFD-HFD, CAF-CAF, HSD-HSD groups). Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 2

Effect of nutritional programming and hypercaloric diet exposure after programming (offspring diet) on food consumption. a and d) Food was weighted daily for 28 days for the different groups both maternal programming and offspring diet. b and e) Kcal per day was calculated by group in this time frame. c and f) Food efficiency by week was calculated. Data are means ± SD. *p ≤ 0.05

Fig. 3

Effect of nutritional programming and offspring diet on glucose homeostasis. a, d, g and j) Basal glucose was measured for all groups. b, e, h and k) GTT and ITT Tests were performed at 15, 30, 45, 60, 90, and 120 min. c and f) Area Under Curve for GTT. i and l) Area Under Curve for ITT. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 4

Lipidomic profile in plasma samples. Lipids were extracted from plasma samples following standard protocols and were analyzed as described in Methods. The concentration of each species was calculated using MultiQuant 3.0.1 software (AB SCIEX, Framingham, MA, USA) by relating the peak area of each species to that of the internal standard. Total plasma TG levels in maternal nutritional programming (a) and maternal and hypercaloric diet exposure after weaning (b). Selective plasma TG species in maternal nutritional programming (c) and maternal and hypercaloric diet exposure after weaning (d). Concentrations are expressed as the mean ± SEM with Chow and HFD, CAF or HSD. n = 10–12. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 5

Effect of nutritional programming and offspring diet on liver and adipose tissue weight. Liver (a, b) and adipose tissue (c, d) were weighted after dissection from all groups. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 6

Effect of nutritional programming and offspring diet on mitochondrial dynamics in hypothalamus. Relative densitometry for protein levels of DRP1 (a, c) or MFN 2 (b, c) n = 8 per group. (c) Pictures are representative of Western Blot for DRP 1, MFN2 and Actin as loading control. mRNA expression levels of MFN2 (d), DRP 1 (e), Opa 1 (f) and Ip3r1 (g) n = 7 per group. Data are means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 7

Effect of nutritional programming of CAF diet plus offspring CAF diet on hypothalamus ultrastructure. a) Control, b) CAF-CAF groups. Note that CAF-CAF diet promote appearance of bigger mitochondria and mitochondria-ER interactions in CAF-CAF (d, 3000×) in compare with Control (c, 3000×). Unstacked and extremely distended endoplasmic reticulum cisternae rims extended (* asterisk) around mitochondria (▼ arrow head) was observed in CAF-CAF group. N, nucleus; G, Golgi apparatus

Fig. 8

Lipotoxicity of palmitic acid induced decrease in mitochondrial mass, membrane potential (ΔΨm) and in ER signal. (a) Representative confocal microscopy images of ΔΨm, which was measured by the retention of TMRM (red), and ER-Tracker Green to define the cytosol. (b) Quantification of mitochondrial membrane potential. Mitochondrial mass, which was calculated from the images stained with TMRM and ER-Tracker Green. ER-Tracker Green fluorescence intensity. Data are mean ± SEM, and values are from three independent experiments. *p < 0.01 compared to control group. Scale bar = 10 μm

Fig. 9

Lipotoxicity of palmitic acid induced changes in mitochondrial Ca²⁺ levels. a) Hypothetical model of calcium over flux from ER to mitochondria and metabolic complications during lipotoxicity. b) Mitochondrial Ca²⁺ after 3 h of treatment with palmitic acid and the normalised values of Rhod-2 AM fluorescence are shown in the histogram. Data are mean ± SEM, and values are from three independent experiments