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High Fat Diet Alters Lactation Outcomes: Possible Involvement of Inflammatory and Serotonergic Pathways

Abstract


Delay in the onset of lactogenesis has been shown to occur in women who are obese, however the mechanism altered within the mammary gland causing the delay remains unknown. Consumption of high fat diets (HFD) has been previously determined to result decreased litters and litter numbers in rodent models due to a decrease in fertility. We examined the effects of feeding a HFD (60% kcal from fat) diet versus a low-fat diet (LFD; 10% kcal from fat) to female Wistar rats on lactation outcomes. Feeding of HFD diet resulted in increased pup weights compared to pups from LFD fed animals for 4 d post-partum. Lactation was delayed in mothers on HFD but they began to produce copious milk volumes beginning 2 d post-partum, and milk yield was similar to LFD by day 3. Mammary glands collected from lactating animals on HFD diet, displayed a disrupted morphologies, with very few and small alveoli. Consistently, there was a significant decrease in the mRNA expression of milk protein genes, glucose transporter 1 (GLUT1) and keratin 5 (K5), a luminobasal cell marker in the mammary glands of HFD lactating animals. Expression of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in serotonin (5-HT) biosynthesis, and the 5-HT7 receptor (HTR7), which regulates mammary gland involution, were significantly increased in mammary glands of HFD animals. Additionally, we saw elevation of the inflammatory markers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α). These results indicate that consumption of HFD impairs mammary parenchymal tissue and impedes its ability to synthesize and secrete milk, possibly through an increase in 5-HT production within the mammary gland leading to an inflammatory process.


Introduction


Obesity has profound negative impacts on numerous physiological processes. Prevalence of obesity of adult women in the United States from 1999–2008 is approximately 36%, with approximately 60% of women of a reproductive age (20–39 years of age) being either overweight or obese [1]. Offspring from obese mothers have consistently displayed negative outcomes such as increased birth weights and increased probability of obesity and metabolic syndrome in their lifetime [2]. Furthermore, excessive weight gain during pregnancy increases the mother's risk of developing breast cancer [3]. While much research has been directed towards examining the impact of maternal obesity on the offspring, little has focused on the mammary gland structure itself. Several studies in humans have demonstrated that obese women have a delay (>72 h post-partum) in the arrival of a copious milk supply [4]–[7]. Delayed lactogenesis (failure to lactate for >72 h post-partum) is correlated with a shorter duration of breast-feeding [8]. Other mammalian species also display lactation defects due to obesity/over-feeding. In fact, in dairy cattle over-feeding during the pre-pubertal period has been determined to cause a permanent decrease in milk yield potential [9]. Furthermore, in studies in rat models, pre-pregnancy overweight and obesity has been determined to decrease the response to suckling induced prolactin and disrupt normal mammary gland development during pregnancy [10]. Additionally, consumption of high-fat diets during pregnancy has also been determined to decrease the amount of myoepithelium in the mammary gland, which is thought to increase breast cancer risk [11].

Several studies have been conducted in different mammalian species demonstrating an association with obesity and mammary development during pregnancy. In a study in obese mice, a delay in lactogenesis appeared to be related to the accumulation of lipid droplets within the epithelial cells, and resulted in decreases in milk protein gene expression [12]. In an experiment conducted in gilts, it was determined that feeding of increased energy during the late gestation period resulted in a 27% decrease of total mammary parenchymal weight compared to gilts fed a diet of adequate energy [13]. Diet-induced obesity has also been determined to negatively impact the stromal compartment of the mammary gland and is associated with an increase in breast cancer risk [11].

Obesity is characterized by the activation of the inflammatory process in metabolically active organs in the body. Typically this response leads to the elevation of pro-inflammatory cytokines, adipokines and other inflammatory makers [14]. Recently it was demonstrated that mice consuming HFD exhibit elevated levels of the monoamine 5-HT in serum [15]. It has been suggested that 5-HT is an important mediator of inflammatory responses, including obesity [15]–[17]. In fact, adipocytes have been determined to synthesize and secrete 5-HT [18]. Additionally, in a mouse model of colitis, an inflammatory disease, animals lacking the rate-limiting enzyme for 5-HT synthesis (TPH1) displayed decreased severity of the disease, along with significantly lower macroscopic and histologic damage scores [17]. When animals were then supplemented with 5-hydroxytryptophan, a precursor to 5-HT synthesis that bypasses TPH1, the severity of the colitis was increased and pro-inflammatory cytokines were induced. Specifically, IL-6 and TNF- α were elevated in animals receiving supplemental 5-hydroxy-L-tryptophan, and were decreased in mice lacking TPH1 [17]. Furthermore, serum 5-HT levels were determined to be elevated in mice consuming HFD [15]. This suggests that 5-HT, along with numerous other factors, is in part responsible for the inflammatory process associated with obesity.

Mammary gland involution, like obesity and colitis, resembles an inflammatory process [19]–[21]. In fact, in several species elevated mRNA expression for IL-6 and TNF-α, among other inflammatory markers, in the mammary gland is typical during the involution process [20]–[21]. Serotonin has been previously demonstrated to be responsible for mammary gland involution in several mammalian species [22]–[25]. In the mammary gland, 5-HT accelerates involution in response to milk stasis by acting on the HTR7 to disrupt tight junctions (TJ), as evidenced by decreases in transepithelial resistance as well as decreased levels of tight junction proteins [23], [25]. Transiently, 5-HT acts to decrease TJ permeability through protein kinase A, and sustained increases in 5-HT results in increased TJ permeability by activation of p38 MAP kinase, thereby initiating mammary gland involution [25].

Currently, little is known about the specific processes that are altered within the mammary gland parenchymal tissue in the obese state and how they contribute to the impaired lactation observed in obese subjects. The objectives of this study were to examine the effects of feeding a HFD on the mammary gland and the ability to lactate. Specifically, we also aimed to delineate the involvement of 5-HT in regulating the mammary gland's response to HFD.


Results


Pre-pregnancy feed intake and body weight gain

HFD animals had significantly higher weekly body weights and body weight gain for the final 3 weeks prior to mating (Figures 1a, b). Cumulative caloric intake was significantly higher in HFD animals during the feeding period pre-mating (Figure 1c). Daily food intake was higher for the first two weeks of feeding in the HFD animals, but did not differ from the LFD animals during the final 4 weeks prior to mating (Figure 1d). Feed intake and body weights were not measured in pregnant and post-partum animals.


Pre-pregnancy body weights and feed intake in female Wistar rats consuming a HFD vs. LFD.

(A) Pre-pregnancy body weights for 6 weeks prior to mating in female Wistar rats consuming a high-fat diet (N = 6; HFD; 60% kcal from fat) versus a low-fat diet (N = 6; LFD; 10% kcal from fat); (B) Pre-pregnancy body weight gain for 6 weeks prior to mating in female Wistar rats consuming a HFD versus a LFD; (C) Pre-pregnancy cumulative food intake for 6 weeks prior to mating in female Wistar rats consuming HFD versus LFD; (D) Pre-pregnancy average daily kilocalorie intake in female Wistar rats consuming HFD versus LFD. Data are represented as mean ± SEM.


Milk production and pup growth rate


Both the control (12±1.4) and HFD (10±1.5) cohorts that successfully gave birth did not have statistically different litter sizes (Figure 2a). HFD animals that successfully carried their pregnancies to term failed to produce any measurable milk yield during the weigh-suckle-weigh test on d1 post-partum, while LFD animals were producing approximately 0.1 g of milk during a bout of nursing on d1 post-partum (Figure 2b). Additionally, we attempted to milk all animals and HFD mothers were unable to produce any milk on d 1 post-partum. We also measured PRL and PRL receptor mRNA concentrations in the mammary glands and did not see any significant differences between cohorts, indicating suckling-induced PRL release was consistent between dams from both the LFD-lactating and HFD-lactating groups (data not shown). Beginning d 2 post-partum, HFD animals began producing milk and the volume did not significantly differ from that of LFD animals through 4d post-partum (Figure 2b). Pups from mothers receiving the HFD were significantly heavier than LFD pups at their first weighing, and for the first 4d post-partum (Figure 2c).


Consumption of HFD alters milk production and pup growth in rats.

(A) Milk weights collected by the weigh-suckle-weigh method in female Wistar rats consuming a high-fat diet (HFD; 60% kcal from fat) versus a low-fat diet (LFD; 10% kcal from fat) for 4 d post-partum. (B) Pup weights measured daily for 4 d postpartum in female Wistar rats consuming a high-fat diet (N = 3; HFD; 60% kcal from fat) versus a low-fat diet (N = 6; LFD; 10% kcal from fat). All data are represented as mean ± SEM (* P<0.05).

Milk lactose was not significantly different between LFD and HFD lactating animals on d 4 of lactation. (Figure 2d). However, milk glucose levels were significantly different between the LFD and HFD cohorts on d 4 lactation (P<0.05; Figure 2e).


Mammary gland histology and alveolar cells


Lactating animals on the HFD displayed disrupted mammary gland morphologies compared to those on the LFD when examined on d 4 lactation (Figure 3). Lactating-HFD mammary gland tissue contained more and larger adipocytes. The alveolar lumens in the parenchymal tissue were smaller, and the alveoli were irregular, with areas in which the parenchymal tissue appeared to have been deteriorating. Additionally, of the alveoli that were intact, they appeared to mostly be empty, and of the ones that did contain fluid in the luminal space, they were more distended than those in the LFD group. Quantitatively, the number of intact alveolar units were significantly reduced in both the HFD-lactating and HFD-non lactating (post-partum) animals compared to LFD-lactating animals (Figure 3b). While it is not surprising that non-lactating animals had few intact alveolar units, the decrease in intact alveolar units in the HFD-lactating animals suggests that obesity altered the development of the alveolar units necessary for milk production.


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