We confirmed the hypothesis that mothers cafeteria diet differently influence metabolic profiles, fat content and inflammation parameters in males and females rat offspring. Besides study of pups, we also examined changes in females before during pregnancy and during lactation. We found that dams on CAF diet had increased food intake in second and third week before pregnancy and in second week of pregnancy when compared to C group. These findings are in agreement with data obtained by Sanchez‑Blanco et al., which shown that female rats kept on CAF diet 22 days before pregnancy and during pregnancy had higher food intake then control 48.
In our study, in spite of reported here increase in food intake in dams, no statistically significant differences in mothers’ weight and fat results were noticed. But, inspection of figures (Figs. 3a and b) indicate a non-significant increase in body weight in dams. These, non-significant effects maybe a result of small study groups (n = 3 mothers/ group). Indeed, studies on bigger number of dams (n = 16/group) shown that 6–13 weeks of exposure to CAF diet rats induced increase in weight in dams 36. Additionally, it was found that CAF rats gained an extra 22% in weight during the pre-pregnancy period compared to controls, and were heavier during pregnancy. Moreover, CAF diet fed dams still remained heavier than animals fed a chow diet at mid-lactation 36.
Our studies also confirmed that both MRI and NMR are useful, non-invasive techniques in assessment of fat contents and body composition, which could be successfully employed in studies of effects of CAF diet on development of obesity. However, one limitation of MRI study in anesthetized animal is heart and breathing movements, which unable to examine abdominal fat. Thus, in this study we could only examined volume of fat tissue on the neck of animals. Here due to logistic considerations, we were not able to perform MRI studies in pups. But, in the future we plan experiments using MRI on pups to examine changes in fat content at different time points of postnatal development.
The main aim of this study was however to examine influence of the CAF diet on pups and study possible sex differences in response to this metabolic insult.
We have found that while on PND 3 there was no difference in body weight between pups on CAF and C diets. Such differences were revealed by PND 25 when both males and females offspring from mothers kept on CAF diet had lower weight compared to control. Additionally, we have found that at PND 25 both pups of both sex from mother kept on CAF diet had higher fat contents. Thus, in our study we have confirmed reported by Pomar et al. 37 thin outside, fat inside phonotype in pups. Moreover, we have revealed that these differences were more pronounced in females offspring.
Cardenas-Perez et al. shown that offspring from mothers kept on CAF diet for 9 weeks (including pre-mating, pregnancy and lactation) had slightly lower body weight at birth comparing to C group 49. Moreover, when offspring were fed CAF diet after weaning, this decrease in body weight persist until week 7 of age. Study performed by Sanchez-Blanco 48 body weight at birth in pups from mothers kept of CAF diet 22 days before pregnancy and during pregnancy was also lower, compared to control. Additionally, when pups remained on CAF diet continue to be lighter until PND 21 48. Similarly, in experiments conducted by Bayol 35 revealed that when CAF diet was given during gestation and lactation to mothers it caused a decrease in body weight of offspring both at birth and at weaning (PND 21). However, when such diet was continuing up to week 10 postnatally an increase in body weigh both in males and females was reported 35. Thus, our data and discussed above studies indicate that CAF diet given to mothers before pregnancy, during pregnancy and lactation leads to decrease in body weight in offspring. Lower body weight of offspring exposed prenatally to CAF diet may be related to decrease protein intake by approximately 37% and 34% during gestation and lactation, respectively 50.
Our study also shown that in CAF females’ offspring at PND 25 decrease in body weight is more pronounced compared to CAF males’ offspring. Sex differences in body weight of offspring was also reported in experiment when mixture of high fat (HFD) and CAF diets (HFD + CAF) were given before mating, and during pregnancy to rats 51. Males but not females’ offspring from HFD + CAF groups had lower weight on PND 1. However, on PND 19 both male and female offspring form mothers on HFD + CAF diet, which remained on such diet after delivery, had higher body weight compared to C group 51.
Numerous experiments including our current study also confirm that exposure to CAF diet: 1) before pregnancy and during pregnancy and lactation, 2) during pregnancy and lactation, 3) only during lactation leads to accumulation of fat tissue in offspring 37,38,52,53.
Sex-specific differences were also found in blood glucose and insulin levels. On PND 25 only in females from CAF mothers was significant increase in blood glucose level. However, in case of insulin, concentration of this hormone was statistically significant higher both in males and females offspring form mother kept on CAF diet. But, again this rise in insulin level was more pronounced in females offspring.
George et al. shown, that both sexes of Wistar rats offspring at age of 12 weeks from mothers kept on CAF diet during pregnancy had not changes in fasting serum glucose levels. However, when dam was exposed to CAF during lactation offspring had significant higher level of fasting serum glucose comparing to control 36. Moreover, only male offspring from mothers kept on CAF diet during lactation had higher pick of glucose in glucose tolerance test at 12 weeks 36. Pomar et al., also shown that offspring at 3 and 6 weeks of age from mothers kept on CAF diet during lactation had impaired response to glucose tolerance test 37. Furthermore, study of George 36 and Pomar 37 found no difference in plasma insulin levels between CAF and C offspring. However, George et al. 36 shown that overall male offspring had significant higher level of plasma insulin compared with female, particularly at baseline and two hours. Thus, the Authors confirm previous finding that exposure during lactation to CAF diet causing male offspring to be more susceptible to insulin resistance 54,55. It was also concluded that only postnatal exposure to maternal obesity lead to adiposity and insulin resistance. It was proposed that the milk composition of the dams is key factor for programming effects. In our study offspring remains on CAF diet while in utero as well as during lactation, which prevent us to dissociate between prenatal and postnatal effects of the diet. Additionally, we have studied offspring in much younger age (PND 25). However, similar to above mentioned studies we also revealed sex-specific effects of CAF diet on metabolic outcomes.
Effects of CAF diet on glucose levels were also studied in offspring. Bayol et al. revealed that exposure of Wistar rats to CAF diet during pregnancy, lactation and post-weaning period leads to sex differences in both glucose and insulin levels in offspring 35. Similar to our results at PND 25, females at age 10 weeks from mother on CAF diet had higher concentration of glucose level when compared to control 35. However, both in Bayol 35 and our studies there was no difference in glucose levels between males offspring form CAF and C mothers. On the other hand, concentration of insulin was significant higher in male offspring in comparison to control, but with no difference in female offspring at age of 10 weeks 35. In our study, with shorter exposure to CAF diet, raised level of this hormone was seen both in male and female offspring. Thus, additional feeding post weaning with CAF diet may contribute to increased levels of insulin observed in males in age of 10 weeks. However, study performed by Bayol 35 also indicate that CAF -induced adiposity and metabolic disruptions were increased in adult offspring from mothers exposed to such diet in pregnancy and lactation when compared with offspring only fed the diet after weaning. Indeed, it was shown that adipocyte hypertrophy and increase in perirenal fat pad mass relative to body weight persist, even when the offspring were fed a chow diet after weaning.
Here we have provided novel finding on sex-specific differences in inflammatory parameters in offspring in response to maternal CAF diet. Significant increase of interleukin 6 (IL-6) and tumor necrosis factor (TNF-α) was found in females from CAF mothers, but not in males offspring. In contrast, significant elevation of interleukin 10 (IL-10) was found in both sexes of offspring. However, concentration of IL-10 in females from CAF mothers was higher than in male offspring. Our results are in agreement with studies performed on adult rats exposed to CAF diet. CAF diet leads to TNF-α overexpression in the intestine 56 and in plasma 33 in adult male rats 5. CAF-fed adult rats also displayed remarkable inflammation in white fat, brown fat and liver 33. Both in animal and human obesity low-level chronic inflammation and macrophage infiltration into adipose tissue is a well-documented phenomenon 57–59. However, signs of inflammation were found not only at the periphery but also centrally, as consumption of CAF diet increases both peripheral and central levels of interleukin-1β (IL-1β), a pro-inflammatory cytokine. Eighteen weeks of administration of Western diet elevated blood serum level of TNF-α and decreased levels of IL-10 and IL-6 in in male Wistar rats, when compared to control 44. However, shorter − 6 weeks of administration of CAF diet to male adult rats had no significant influence of diet on inflammatory parameters (TNF-α and IL-6) 34. But, longer administration of CAF diet (15 weeks) to adult mice leaded to inflammatory processes, characterized by significant higher concentration of IL-6 25. Thus, duration of administration of CAF diet maybe a key parameters when discussing inflammatory responses. In contrast to well documented inflammation caused by CAF diet in adult animals, literature on this subject in prenatal models is spare. When CAF diet was given to rat mothers 8 weeks before pregnancy and during pregnancy it did not increase the inflammatory status of the mother, placenta or fetus in late gestation (PND 21) 45. Actually, levels of inflammatory markers such as IL-6, IL-12p40 and MIP2 were reduced slightly. Carillon et al. shown that Wistar rats offspring from mothers kept on CAF diet for 9 weeks, including pregnancy and lactation had higher mRNA expression of IL-6 in brain than C. However, differences in TNF-α mRNA expression was not observed 23.
In spite of that CAF diet is commonly used in many laboratories, the main caveat refers to its nutritional composition, which is uncontrolled, given that the animals can choose among a variety of different foods 60. There is no standard CAF diet protocol, its caloric content and selection of products varies largely between studies 61. Additionally, percentage of energy derived from fat and carbohydrates differs and ranges between 17–60 and 37 and 73, respectively. Moreover, while in some studies CAF was used alone, in other it has been combined with standard chow diet. However, one way to overcome these difficulties is to simplified the diet, and employed with only 3 or 4 food choices 62,63, which were used in the current study. Finally, protocols used by different researches varied in time of exposure to the diet.
In summary, we have shown that mothers exposure to CAF diet, which mimic “Western-style diet” before and during pregnancy and lactation increased body weight, fat contents, changed metabolic profiles (levels of glucose and insulin), and led to inflammation in offspring. Moreover, we have revealed that above alterations are sex-specific and more pronounced in female offspring. We have also shown that non-invasive imaging techniques such as NMR and MRI could be successfully employed in CAF-induced obesity model. Our study indicates a crucial need to monitor early sign of development of metabolic problem in offspring form mothers kept on CAF diet with respect to sex differences.