Cigarette Smoke During Breastfeeding in Rats Changes Glucocorticoid and Vitamin D Status in Obese Adult Offspring

Maternal smoking increases obesogenesis in the progeny. Obesity is associated with several hormonal dysfunctions. In a rat model of postnatal tobacco smoke exposure, we previously reported increased central fat depot and disruption of some hormonal systems in the adult offspring. As both glucocorticoids and vitamin D alter lipogenesis and adipogenesis, here we evaluated the metabolism of these two hormones in visceral adipose tissue (VAT) and liver by Western blotting, and possible associations with lipogenesis biomarkers in adult rats that were exposed to tobacco smoke during their suckling period. At postnatal day (PN) 3, dams and offspring of both sexes were exposed (S group) or not (C group) to tobacco smoke, 4 × 1 h/day. At PN180, corticosteronemia was lower in S male and higher in S female offspring, without alterations in peripheral glucocorticoid metabolism and receptor. Adrenal ACTH receptor (MC2R) was higher in both sexes of S group. Despite unchanged serum vitamin D, liver 25-hydroxylase was higher in both sexes of S group. Male S offspring had higher 1α-hydroxylase, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) in VAT. Both sexes showed increased ACC protein content and reduced sirtuin mRNA in liver. Male S offspring had lower liver peroxisome proliferator-activated receptor-α. Tobacco exposure during lactation induced abdominal obesity in both sexes via distinct mechanisms. Males and females seem to develop HPA-axis dysfunction instead of changes in glucocorticoid metabolism and action. Lipogenesis in VAT and liver, as well as vitamin D status, are more influenced by postnatal smoke exposure in male than in female adult rat offspring.


Introduction
It is well established that smoking is a risk factor for the development of some chronic non-communicable diseases, such as respiratory diseases, some types of cancers, cardiovascular diseases, and glucose intolerance, in addition to being one of the major causes of early death show higher serum 25(OH)D levels associated with increased fat mass [36,37]. Furthermore, higher 1-alpha-hydroxylase (the enzyme that converts 25(OH)D to 1,25(OH)2D) in the visceral adipocyte and a lower VDR, have been reported, suggesting vitamin D resistance in the adipocyte [36,37]. Thus, the absence of vitamin D action could be obesogenic. Concerning liver tissue, epidemiological studies have identified an association between low levels of vitamin D and NAFLD [38,39]. In animals, vitamin D improves hepatic steatosis by preventing autophagy and oxidative stress [40,41], but the mechanism by which vitamin D attenuates NAFLD remains unclear.
Based on the effects of glucocorticoids and vitamin D on lipogenesis and on the phenotype shown by adult animals of both sexes that were programmed by neonatal exposure to cigarette smoke, the objective of the present study was to evaluate the metabolism of these two hormones and the their relationship with lipogenic markers in adipose tissue and liver of rats that were tobacco smoke-exposed from birth to weaning. We hypothesized that the metabolism and/or action of glucocorticoids and vitamin D in adipocytes and hepatocytes would be altered, and that these alterations could contribute to obesogenesis in these animals. Additionally, because sirtuin 1, a histone deacetylase that is associated with lower adiposity and protection against metabolic disorders [42,43], is inhibited by smoking [44], we also evaluated its mRNA expression in the liver to further elucidate possible molecular mechanisms. Figure 1A,B and Figure 2 depict the morphology of retroperitoneal adipocytes and liver, respectively. C and S groups of both sexes showed preserved architectures of these tissues. Figure 1C,D show that visceral adipocyte size did not differ between the groups. According to the Brunt's NAFLD score, hepatic histological evaluation of males and females of both groups showed grade 0, which means that <5% of hepatocytes were affected ( Table 1). As shown in Table 2, there were no differences between S and C groups of both sexes regarding triglyceride and cholesterol levels in the liver. Groups: C-CONTROL and S-SMOKE; n = 5-6 per group.

Figure 2.
Representative photomicrographs (×60; hematoxylin-eosin) of the liver of adult rats programmed by smoke exposure during the lactation period. (A) morphology of C and S males; (B) morphology of C and S females. Both groups presented typical architecture, with no inflammatory cell infiltrate or drops of lipids in their cytoplasm. Groups: C-CONTROL and S-SMOKE (n = 6 rats/group).

Serum Corticosterone and Adrenal ACTH Receptor (MC2R).
Male offspring of the S group showed lower corticosteronemia (−33% vs. C, p < 0.05, Figure 3A) and higher MC2R content in the adrenal gland (+52% vs. C, p < 0.05, Figure 3C), while S females had higher corticosteronemia and higher MC2R content (+1.5-fold vs. C, p < 0.05, Figure 3B; +1.3-fold vs. C, p < 0.05, Figure 3D; respectively).  Figure 4 depicts the content of the enzyme that generates corticosterone (11β-HSD1) and the glucocorticoid receptor (GRα) in the VAT and liver. There were no differences between S and C groups in the adult offspring of both sexes (males: Figure 4A,B,E,F; females: Figure 4C,D,G,H) concerning these two glucocorticoids biomarkers on both tissues.  Figure 4 depicts the content of the enzyme that generates corticosterone (11β-HSD1) and the glucocorticoid receptor (GRα) in the VAT and liver. There were no differences between S and C groups in the adult offspring of both sexes (males: Figure 4A,B,E,F; females: Figure 4C,D,G,H) concerning these two glucocorticoids biomarkers on both tissues.    Figure 5 shows: (a) the content of the enzyme that generates calcidiol (25-hydroxylase) in the liver, (b) the calcidiol levels in the serum, and c) the content of the enzyme that activates the calcitriol in the VAT (1α-hydroxylase). Male offspring of the S group had higher 25-hydroxylase protein expression (+1.3-fold vs. C, p < 0.05, Figure 5A), unchanged 25 (OH) D levels ( Figure 5B), and higher 1α-hydroxylase (+94% vs. C, p < 0.05, Figure 5C). Females of the S group had higher 25-hydroxylase in the liver (+97% vs. C, p < 0.05, Figure 5D), but no alterations in serum vitamin D and VAT 1α-hydroxylase ( Figure 5E,F).  Figure 5 shows: (a) the content of the enzyme that generates calcidiol (25-hydroxylase) in the liver, (b) the calcidiol levels in the serum, and c) the content of the enzyme that activates the calcitriol in the VAT (1α-hydroxylase). Male offspring of the S group had higher 25-hydroxylase protein expression (+1.3-fold vs. C, p < 0.05, Figure 5A), unchanged 25 (OH) D levels ( Figure 5B), and higher 1α-hydroxylase (+94% vs. C, p < 0.05, Figure 5C). Females of the S group had higher 25-hydroxylase in the liver (+97% vs. C, p < 0.05, Figure 5D), but no alterations in serum vitamin D and VAT 1αhydroxylase ( Figure 5E,F).

Lipogenesis in Visceral Adipose Tissue
The contents of the proteins related to lipogenesis (ACC and FAS) are shown in Figure 6. We found higher ACC (+1.0-fold vs. male C p < 0.05, Figure 6A) and FAS (+1.3-fold vs. male C p < 0.05,

Lipogenesis in Visceral Adipose Tissue
The contents of the proteins related to lipogenesis (ACC and FAS) are shown in Figure 6. We found higher ACC (+1.0-fold vs. male C p < 0.05, Figure 6A) and FAS (+1.3-fold vs. male C p < 0.05, Figure 6B) protein contents in the males of the S group when compared to controls. Females did not show differences between groups regarding ACC and FAS in the VAT ( Figure 6C,D, respectively).  Figure 6B) protein contents in the males of the S group when compared to controls. Females did not show differences between groups regarding ACC and FAS in the VAT ( Figure 6C,D, respectively).

Discussion
In the present study, adult animals that were exposed to cigarette smoke during their breastfeeding period showed changes in serum corticosterone and tissue metabolism of vitamin D

Discussion
In the present study, adult animals that were exposed to cigarette smoke during their breastfeeding period showed changes in serum corticosterone and tissue metabolism of vitamin D that may contribute to adipose tissue and liver dysfunction, although the morphology of both tissues was normal. Sexual dimorphisms were observed regarding circulating hormones and metabolic changes at the tissue level.
Recently, in an animal model of exposure to neonatal cigarette smoke, Lisboa et al. (2017) showed that adult animals of both sexes present increased abdominal adiposity. In the current study, these results were also observed [17]. However, when evaluating the morphology of VAT (retroperitoneal depot) and liver in these animals, surprisingly, no alteration of area or lipid accumulation was observed. Our study corroborates that of Zinkhan et al. (2014), in which exposure to smoke during pregnancy also increased adiposity in adult males without changes in adipocyte area [45]. Thus, here we suggest that the increase in adiposity observed in adult animals may be due to an increase in adipogenesis, indicating that this obesity is hyperplastic, not hypertrophic. We also did not detect changes in the contents of cholesterol and triglyceride in the liver, despite the several changes that were observed in lipogenic markers in the liver. In the literature, there are no data on the morphology of the hepatic tissue of animals exposed to cigarette smoke during the lactation period. In adolescent mice, chronic exposure to cigarette smoke does not alter the morphology of this tissue or the expression of genes that regulate lipid metabolism, inflammatory markers, and genes related to hepatic fibrosis [46].
The role of glucocorticoids as regulators of lipid accumulation is already well established; it acts by stimulating the adipocyte differentiation as well as the distribution of body fat [47]. In the current study, we observed alterations in corticosteronemia, with males showing a reduction and females an increase, which is indicative of a sexual dimorphism regarding tobacco smoke action regarding this parameter. To better understand this finding, we investigated MC2R in the adrenal, and glucocorticoid tissue metabolism (11β-HSD1 and GRα) in the VAT and liver. An increase of MC2R was observed in both sexes, without alterations in either GRα or 11β-HSD1 contents in the two tissues. This finding indicates that some alteration in the HPA (hypothalamus-pituitary-adrenal) axis secretion is present. The reduction in corticosteronemia seen in males may be due to primary adrenal hypofunction at adulthood, characterized by increased CRH-ACTH, and may be related to the hypercorticosteronemia of these animals during early life, as previously demonstrated [16]. An increase in ACTH secretion by the pituitary gland could overstimulate its receptors in the zona fasciculata of the adrenal gland, since it has been described that MC2R shows the unusual characteristic of being up-regulated by its ligand [48,49]. Conversely, females exposed to smoke were programmed for hypercorticosteronemia with increased MC2R, indicating a possible Cushing's disease (ACTH dependent). Contrasting with males, females at the beginning of life did not present alterations in corticosteronemia [17]. Thus, we suggest that females exposed to smoke appear to be protected from its immediate effects, but not from adrenal function programming, by some still unknown mechanism. Another possibility is that cigarette smoke may directly increase CRH-ACTH axis or MC2R. However, Zinkhan et al. [45] demonstrated that intrauterine exposure to tobacco causes hypercorticosteronemia in the offspring of adult males (60 days), while hypocorticosteronemia is observed in females. The main difference between the aforementioned study and our present one is the period of smoke imprinting (gestation vs. lactation).
Some studies have demonstrated additional bone actions of vitamin D; among them is its action on lipid metabolism, which may directly influence the development of obesity [27,30]. However, the relationship between vitamin D and obesity is still quite controversial. Obese human subjects show reduced serum vitamin D levels [50,51], whereas in obese rodents, no change in serum levels was found [46]. In different experimental models of programming for obesity, an increase in 25 (OH) D concentrations was observed. Thus, it is possible that both the deficiency of and the resistance to this hormone are involved in the obesogenesis. For this reason, we evaluated the vitamin D status of adult animals programmed by the direct exposure to cigarette smoke during the lactation period. Despite no alteration in 25 (OH) D serum levels, both sexes showed increased hepatic 25-hydroxylase. It is possible that cigarette-smoke compounds may affect the absorption of vitamin D precursors, but the increase in liver 25-hydroxylase corrects the calcidiol serum levels. Also, this hormone may be highly activated to 1.25 (OH) D in the kidney by 1α-hydroxylase or inactivated to 24.25 (OH) D by 24 hydroxylase. In obesity, it has been suggested that the reduction in circulating vitamin D levels is due to a greater sequestration by adipocytes. The male offspring showed higher 1α-hydroxylase in the visceral adipocyte, which may indicate a greater uptake, and consequently, a higher local vitamin D formation. The increase in VAT 1α-hydroxylase has already been observed in other models of obesity programming, i.e., those in which a reduction of VDR is also present, suggesting a resistance to vitamin D action [37,52]. Thus, since vitamin D has anti-adipogenic effects [31][32][33], it is possible that our animals, which were programmed by the exposure to tobacco during the lactation period, developed resistance to vitamin D in the adipocyte, which contributes to their hyperplasia. Unlike males, the metabolism of vitamin D in the adipocyte of females does not seem to contribute to the increase in their adipocyte mass, characterizing a sex-dependent effect.
In order to understand the increase in central adiposity in our model, we analyzed the lipid metabolism in both sexes and, curiously, we observed alterations only in males, with increases in ACC and FAS, enzymes that are responsible for the synthesis of triglycerides and fat acids [53,54]. The increase in triglycerides in the adipose tissue is indicative of cellular dysfunction, and may trigger cellular apoptosis [54]. The results in the male adipose tissue suggest an increase in triacylglycerol production and, as a consequence, an increase in central adiposity. As females do not have alterations in these enzymes, we suggest that this mechanism is sex-dependent. Our results corroborate the study of Fan et al. (2016), which used a model of exposure of mothers dams and pups to nicotine during the prenatal and postnatal periods. They demonstrated increased central adiposity and FAS in males, while females also showed increased central adiposity but without alterations in FAS activity, suggesting a deleterious effect of nicotine programming on lipogenesis, with sex-related differences. While we realize that cigarette smoke has thousands of substances, we think that the changes described in our study are directly related to the effects of nicotine, mainly due to the results observed by Fan et al. [55].
Although we did not observe important alterations in the liver tissue under optical microscopy, we analyzed transcription factors and proteins involved in lipid metabolism: Animals presented an imbalance in hepatic lipid metabolism, with reduced markers related to lipid-β-oxidation and increased markers related to lipogenesis. The relationship between smoking, mainly through the actions of nicotine, and sirtuin, a histone deacetylase (HDAC), has already been established. For example, exposure to cigarette smoke reduces Sirt1 levels in vitro and in vivo, with associated increases in the release of proinflammatory cytokines [44,56]. In addition, there is a direct relationship between sirtuins and PPARs: Sirt1 activates PPARα through the deacetylation of PGC-1α [57,58], inhibiting its transcription. In the present study, males had a reduction in both Sirt1 and PPARα, which is indicative of the mechanism through which lipid accumulation occurs in the liver, and that may contribute to the genesis of hepatic steatosis. Corroborating this idea of hepatic dysfunction, we observed increased ACC in the male offspring. Regarding females, we observed a similar profile in the analyzed markers when compared to males, although liver PPARα was unaffected. It is important to emphasize that they also show increased ACC.

Animal Research
This study was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (CEUA/018/2014; 28 January 2014) and was performed in accordance with the principles adopted by Brazilian Law no. 11.794/2008 (8 October 2008). Wistar rats were appropriately allocated in a temperature-controlled room (23-24 • C) with artificial light and dark cycles (lights on: 07.00 h; lights off: 19.00 h).
For mating, 3-month-old virgin female rats were kept with male rats in a 2:1 proportion for 1 week. After mating, pregnant females were placed in individual cages with free access to water and standard chow (Nuvilab, Sogorb, São Paulo, SP, Brazil) during gestation and breastfeeding. At birth, litters were adjusted to 6 rats (3 females and 3 males) per dam.

Experimental Model of Direct Tobacco Smoke Exposure during Suckling Period
Three days after birth, rat dams and their litters were randomly assigned into one of the following groups: (a) SMOKE (S, n = 10): dams and offspring of both sexes were placed into a smoking machine (TE-10, Teague Enterprises, CA, USA), 4 1-h exposures times per day (1 h for each exposure). The tobacco smoke used was obtained from burning one cigarette type 3R4F at a time (nicotine = 0.73 mg/cigt; Reference Cigarette Program, University of Kentucky, Lexington, KY, USA) [16,17,59]. (b) CONTROL (C, n = 10): dams and offspring of both sexes were exposed to ambient air in a chamber that was similar to the one used for tobacco smoke exposure. The experimental cigarettes used in the present study produce cotinine levels in dams and pups at the end of lactation period of, respectively, 105 ng/mL and 145 ng/mL [16], which are similar to those observed in typical (heavy) smokers [60]. After weaning, body mass and food intake were monitored every 4 days until 180 days.

Histology
Fragments of the visceral (retroperitoneal) adipose tissue (retroperitoneal) and liver obtained from all lobes were fixed in freshly prepared fixative (1.27 mol/L formaldehyde, 0.1 mol/L phosphate buffer, pH 7.2) for 48 h at room temperature. Histological processing was carried out with alcohol baths, xylol and paraffin. Tissues were embedded in Paraplast Plus (Sigma-Aldrich, St. Louis, MO, USA) to obtain random cuts with a thickness of 5 µm. These were then stained with hematoxylin-eosin (HE). Adipocyte size was determined using the software Image-Pro Plus, version 5.0 (Media Cybernetics, Silverspring, MD, USA), with the use of randomly acquired digital images (TIFF format, 36-bit color, 1360 × 1024 pixels, 40× magnification) using an Olympus BX40 microscope with an Olympus DP71 camera (Olympus, Tokyo, Japan). A total of 50 adipocytes per animal (n = 8 offspring per sex/group) were measured, totaling 400 adipocytes per group.

Hepatic Determination of Triglyceride and Cholesterol Contents
Liver samples (50 mg) were homogenized in 1 mL isopropanol (Vetec, Rio de Janeiro, Brazil) and centrifuged (2000× g, 10 min, 4 • C). We used n = 10 offspring per sex/group. The total triglyceride and cholesterol levels were measured using a colorimetric method with a commercial kit (Bioclin, Belo Horizonte, Brazil), as previously reported [62].

Serum Hormones Analysis-Radioimmunoassay (RIA) and Electrochemiluminescence Immunoassay (ECLIA)
In both assays, samples were analyzed in duplicate (n = 10 offspring per sex per group). Corticosterone levels were evaluated by RIA kit (ImmuChem TM 125I, coated tube, ICN Biomedicals Inc., New York, NY, USA) following the manufacturer's instructions. The 25(OH)D was quantified using a commercial ECLIA kit (Elecsys Vitamin D total assay, Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacturer's instructions. This hormone is generally used to determine overall vitamin D status [63].

Western Blotting
In the visceral adipose tissue and liver, we evaluated the protein contents of the glucocorticoid receptor α (GRα), 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). In the visceral adipose tissue, we also determined the 1α-hydroxylase (CYP27B1) content. In the liver, we also further measured vitamin D 25-hydroxylase (CYP2R1) and peroxisome proliferator-activated receptor alpha (PPAR-α) contents. In the adrenal gland, we evaluated the melanocortin receptor (MC2R) content, which is an ACTH receptor. In all tissues, we used n = 7 offspring per sex/group.

Sirtuin 1 (Sirt1) mRNA Expression in the Liver
Tissues were stored in RNAlater (Qiagen, Valencia, CA, USA) at −80 • C, to avoid RNA degradation, until the moment of mRNA extraction. Total RNA from the same amount of each sample (50 mg) was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), under RNAse-free conditions. Before cDNA confection, the quantity and the quality of the RNA in each sample were evaluated using the NanoVueTMPlus Spectrophotometer (GE Healthcare, Buckinghamshire, UK). Only the samples that had 260/280 ratio between 1.8-2.0 and the 260/230 ratio between 1.8-2.2 were used on in the RT-PCR assays. The cDNA was constructed from 1 µg of total RNA using the Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) for RT-PCR and Oligo(dT)15 Primer (Promega, Madison, WI, USA). Before the RT-PCR assay, the amplification efficiency of each probe was analyzed by the standard curve and the appropriate threshold and cDNA dilution were determined. The mRNA expression was analyzed by RT-PCR carried out in duplicate for each sample using an Applied Biosystems 7500 Real-Time PCR System (Applied BioSystems, Foster City, CA, USA). To ensure the absence of genomic DNA contamination, a minus RT reaction (RT-) was performed in all RT-PCR assays: no amplification product (Cq value) was detected in any of the RT-control reactions. The expression of Sirt1 (Rn01428096_m1) mRNA was quantified using TaqMan ® Fast Universal PCR Master Mix (2×) AmpErase ® UNG (Catalog #: 4324018) (Applied Biosystems ® , Foster City, CA, USA) in accordance with the recommendations of the manufacturer. A co-amplification of Actin-beta gene (Assay ID: Rn00667869_m1) was also performed in all samples. This gene was chosen as a reference since both the Control and Smoke groups exhibited similar Cq values. Results were analyzed using the ∆∆C T method. We used from 5 to 7 offspring per sex/group.

Statistical Analysis
The statistical analyses were carried out using the Graph Pad Prism 5.0 for Windows statistical software (GraphPad Software, La Jolla, CA, USA). Comparisons between groups were performed using Student's unpaired t-test because the metabolic programming effect was evaluated separately for males and females. Data are shown as means and standard error of the mean (S.E.M.). Differences were considered significant at p < 0.05.

Conclusions
Here, we evidenced that the hormonal and functional outcomes at adulthood of exposure to cigarette smoke exclusively during the lactation period are sex-dependent, being less severe in females. We suggest that sex steroid hormones may influence these differences in response: While testosterone enhances the deleterious effects of programming via exposure to cigarette smoke, estrogen may actually protect females from this endocrine disruptor in order to ensure normal reproduction.