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Does bromocriptine play a role in decreasing oxidative stress for early weaned programmed obesity?
Life Sciences 95 (2014) 14–21
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Does bromocriptine play a role in decreasing oxidative stress for early weaned programmed obesity? Nayara Peixoto-Silva a, Ellen P.S. Conceição a, Janaine C. Carvalho a, Natália S. Lima a, José Firmino Nogueira-Neto b, Elaine de Oliveira a, Egberto G. Moura a, Patricia C. Lisboa a,⁎ a b
Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, Brazil Laboratory of Lipids, School of Medicine, State University of Rio de Janeiro, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 1 August 2013 Accepted 6 December 2013 Keywords: Early weaning Bromocriptine Obesity Liver disease Antioxidant system
a b s t r a c t Aims: Studies have demonstrated that early weaning can promote metabolic syndrome during adulthood and that obesity increases oxidative stress. Thus, we aimed to evaluate redox status in a pharmacological early weaning rodent model programmed for metabolic syndrome at adulthood. Main methods: Lactating dams were randomly assigned into 2 groups: the early weaning group (BRO), which was treated intraperitoneally with bromocriptine (1 mg/day) to inhibit prolactin secretion for the last 3 days of lactation, and the control group (C), which received the BRO diluent for the same time period. The offspring were killed at 90 (PN90) and 180 (PN180) days after birth. Key findings: Early weaning induced greater visceral adiposity and dyslipidemia. At PN90, the BRO offspring showed glucose intolerance with normoinsulinemia and increased plasma and liver superoxide dismutase, and liver glutathione peroxidase activities, which reduced the liver malondialdehyde but not the increased plasma malondialdehyde levels. However, the BRO offspring showed insulin resistance at PN180 and increased plasma glutathione peroxidase, liver superoxide dismutase, and catalase activities. These changes reduced the plasma and liver malondialdehyde levels, which aided in hepatocyte architecture preservation. Additionally, we observed that sirtuin 1 was overexpressed in the BRO group at PN90, but the increased expression was not maintained through PN180, which suggests unfavorable metabolic conditions in the older offspring. Significance: Despite the observed obesity and glucose homeostasis dysfunction, our data suggest that the early weaning programming induced by bromocriptine can improve the offspring's redox status and may prevent liver damage during adulthood. © 2013 Elsevier Inc. All rights reserved.
Introduction Breastfeeding is associated with reduced obesity risk because maternal milk causes hypoinsulinemia, which decreases fat storage and prevents excessive early adipogenesis (Oddy, 2012). In fact, early weaning is related to childhood obesity, which demonstrates that the timing for introducing solid food to an infant's diet affects weight gain (Sloan et al., 2008). Studies were performed to understand the mechanisms involved in the later effects triggered by early weaning. When pups were weaned through maternal separation, the offspring showed a preference for high-calorie food during adulthood, which led to obesity and metabolic changes (dos Santos et al., 2011). Additional alterations were observed during adulthood after early weaning from lactation by applying a ⁎ Corresponding author at: Departamento de Ciências Fisiológicas, 5° andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ, 20551–030, Brazil. Tel.: +55 21 28688334; fax: +55 21 28688029. E-mail address: [email protected] (P.C. Lisboa). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.013
bandage on the mother's body to interrupt lactation without maternal separation. The adult offspring exhibited obesity, dyslipidemia, and hyperglycemia, which are characteristics of metabolic syndrome, hyperleptinemia, central leptin resistance, and higher catecholamine (Lima Nda et al., 2011; Lima et al., 2013). Further, milk production can be suppressed in a pharmacological model for early weaning using bromocriptine, which is a type 2 dopaminergic receptor agonist that inhibits prolactin. Offspring with mothers treated with bromocriptine exhibited neonatal malnutrition and developed obesity, hyperleptinemia, leptin and insulin resistance, dyslipidemia, hypothyroidism, and higher adrenal hormones during adulthood (Bonomo et al., 2007, 2008; de Moura et al., 2009). Long-lasting dysfunction induced by early weaning may lead to liver changes; the liver is a central metabolic organ that regulates energy homeostasis. In nonalcoholic fatty liver disease, metabolic syndrome is manifested; in insulin resistance, oxidative stress and inflammatory cascades can be important for hepatic disease pathogenesis and progression (Colak et al., 2011). Thus, oxidative stress is an important mechanism that links obesity and its comorbidities because both a
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suppressed antioxidant system and an enhanced reactive oxygen species production are related to central adiposity in obese individuals (Savini et al., 2013). Recently, a non-pharmacological early weaning model was programmed for increased oxidative stress and steatosis in the liver, which was reversed through resveratrol treatment. Resveratrol is a natural phytoalexin in grapes, which produces beneficial effects because it is an antioxidant agent (Franco et al., 2013) that activates histone deacetylases, sirtuin 1 (SIRT1), and aids in treating or preventing obesity. Notably, (i) lactation is critical for development, and (ii) a pharmacological early weaning model induces several disruptions in adult progeny that are similar to the disruptions observed in a non-pharmacological early weaning model, such as metabolic syndrome. Therefore, the study herein was designed to evaluate glucose homeostasis, redox status, and hepatic morphological alterations at 2 different time points in adult offspring whose mothers received bromocriptine at the end of lactation. For insight into the programming mechanisms, liver SIRT1 expression was measured in our study. SIRT1 is important for glucose and lipid metabolism (Guarente, 2006), and higher SIRT1 activity has been related to lower adiposity and protection against diet-induced metabolic disorders (Pfluger et al., 2008); therefore, we reasoned that SIRT1 expression may be decreased by early weaning.
Material and methods Ethical approval The experiment was designed and performed in accordance with the principles adopted and promulgated by Brazilian law no. 11.794/2008, and each procedure was approved by the Animal Care and Use Committee at the Biology Institute in the State University of Rio de Janeiro (CEUA/186/2007, CEUA/017/2009). The experiment was performed to minimize the number of animals used and the suffering from the procedures following the three ‘R’s' of ethical doctrine: reduction, refinement, and replacement (Drummond, 2009).
Animals The animals were maintained in a temperature-controlled room (23/24 °C) with artificial dark–light cycles (lights on for 7 h and lights off for 19 h). Three-month-old virgin female Wistar rats were mated with male breeders at a 2:1 ratio. The pregnant rats were maintained in individual cages with free access to water and standard chow until delivery and during lactation. To avoid the influence of litter size on the programming effect, only the mothers with 10 pups were used. After spontaneous delivery, the litters were adjusted to 6 male pups by the mother (anogenital distance was used to determine the gender). Manipulating the litter standardized the food supply and maximized the lactation performance. Beginning at birth, the body mass (BM) and naso-anal length (NAL) were measured in the male pups. The lactating dams were randomly assigned to 2 groups: (BRO, n = 5), which was administered 2 doses of 0.5 mg bromocriptine (Novartis, SP, Brazil) per day (8:00 AM and 8:00 PM) diluted in 200 μL of methanol–saline (1:1 v/v) and injected intraperitoneally for the last 3 days of lactation, and the control group (C, n = 5), which received only a methanol–saline treatment for the same time period. After weaning, 1 pup was randomly selected from each litter (5 animals per group) and analyzed 90 days after birth (PN90). The analyses at 180 days after birth (PN180) were performed using the average between 2 randomly selected pups from each litter, also totaling 5 animals per group.
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Biometric parameters and food intake During lactation, the BM and NAL were measured every 3 days; after weaning, the absolute values for the parameters were measured weekly with the food intake. The food consumption was measured as the difference between the amount of food provided and remaining 7 days later divided by the number of animals in the cage. Oral glucose tolerance test An oral glucose tolerance test (OGTT) was performed at PN90 and PN180. After a 12 hour fasting period, 50% glucose was administered in sterile saline (0.9% NaCl) through an oral gavage at 2 g/kg BM. Blood was drawn from the tip tail of each animal to measure the plasma glucose concentration, which was assessed using a glucometer (Accu-Chek Advantage; Roche Diagnostics, Mannheim, Germany) before the glucose was administered and 15, 30, 60, and 120 min after the gavage (Peixoto-Silva et al., 2011). Euthanasia After fasting for 12 h, the adult rats were sacrificed by quick decapitation to collect the blood, visceral fat mass ([VFM], retroperitoneal, epididymal, and mesenteric fat) and liver. The blood was previously collected in a heparinized tube and centrifuged (2500 rpm, 25 min, 4 °C). The plasma was collected and stored individually at −20 ° C until it was used for the different measurements. The hydrostatic liver weight/ volume was measured using the Scherle method and normalized to the right tibia length in accordance with previous studies (Conceição et al., 2013a,b). The tissues were fractionated and stored using multiple procedures (freezing at −80 °C or fixative solution). Plasma insulin and biochemical analyses The insulin concentration was determined using an RIA kit (ICN Pharmaceuticals, Inc., Orangeburg, NY, USA) with the assay sensitivity 0.1 ng/mL and a 4.1% intra-assay variation. The measurements were performed using a single assay. To determine the adult animals' insulin sensitivity, we used the homeostatic model assessment of insulin resistance (HOMA-IR): (fasting glycemia [mmol/L] × fasting insulinemia [μU/mL] / 22.5). We analyzed the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglyceride (TAG) plasma levels using Biosystem commercial test kits and a spectrophotometer (Biosystems S.A., Barcelona, Spain). Histological processing and adipocyte morphometry Fragments from the retroperitoneal fat and liver were collected from each lobe and fixed in a freshly prepared fixative (1.27 M—formaldehyde, Table 1 Biometry and adiposity of rats whose were early weaned. C BM (g) Birth 18 days old 19 days old 20 days old 21 days old PN90 PN180 VFM (g) PN90 (g) PN180 (g)
6.125 38.43 41.07 43.89 46.62 323.8 398.2
BRO ± ± ± ± ± ± ±
0.08 0.61 0.69 0.62 0.61 9.08 14.03
7.82 ± 0.33 9.44 ± 0.96
5.955 38.05 39.91 41.52 42.97 331.6 421.0
± ± ± ± ± ± ±
0.09 0.79 0.79 0.87⁎ 0.98⁎ 20.24 15.99
11.40 ± 1.51⁎ 13.87 ± 1.64⁎
Values are mean and SEM. P b 0.05; n = 5 per group. C —control offspring; BRO —earlyweaned offspring; BM —body mass; VFM —visceral fat mass. ⁎ significantly different from control offspring.
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Fig. 1. Mean cross-sectional area of adipocytes. Photomicrographs of the retroperitoneal fat with same magnification (×40) and stained with hematoxylin–eosin (HE) demonstrating a hypertrophy of adipocytes in the BRO offspring at PN90 and PN180. C —control offspring; BRO —early-weaned offspring. Values are means and SEM. P b 0.05. n = 5 per group.
0.1 M—phosphate buffer, pH 7.2) at room temperature for 48 h. After histological processing, the tissues were embedded in Paraplast Plus ® (Sigma-Aldrich, St. Louis, MO, USA) to produce random 5 μm thick cuts and stained with hematoxylin–eosin (HE) for visualization using light microscopy.
The adipocyte sectional areas were analyzed using Image-Pro Plus software, version 5.0 (Media Cybernetics, Silver Spring, MD, USA) with randomly acquired digital images (TIFF format, 36-bit color, 1360 × 1024 pixels, 40 ×) using an Olympus BX40 microscope with an Olympus DP71 and camera (Olympus, Tokyo, Japan). In total, 50
Table 2 Metabolic profile of rats whose were early weaned at PN90 and PN180. PN90
Fasting glucose (mg/dL) Range OGTT (AUC) Range Serum insulin (μUI/mL) Range HOMA-IR Range TAG (mg/dL) Range
PN180
C
BRO
C
BRO
70 ± 1.59 64.0–73.0 12,200 ± 267.1 11,408–12,990 31.01 ± 1.87 25.05–36.8 5.38 ± 0.32 4.263–6127 51.0 ± 3.35 42.0–58.0
69 ± 2.65 61.0–77.0 13,460 ± 323.5⁎ 12,180–13,950 35.24 ± 2.66 27.01–43.11 6.01 ± 0.61 4731–8189 61.8 ± 3.97 55.0–76.0
74 ± 4.92 64.5–92.0 14,020 ± 338.0 11,468–15,210 35.79 ± 2.97 25.95–43.04 5.94 ± 0.46 4.75−7.48 55.8 ± 2.35 47.0–60.0
68 ± 2.11 60.5–72.0 14,000 ± 373.6 12,398–17,055 45.57 ± 2.43⁎ 40.71–54.43 7.77 ± 0.56⁎ 6.21–9.50 85.1 ± 10.79⁎ 50.0–113.0
Values are mean and SEM. P b 0.05; n = 5 per group. C —control offspring; BRO —early-weaned offspring; OGTT —oral glucose tolerance test; AUC —area under curve; HOMA-IR —homeostatic model assessment of insulin resistance; TAG— triglycerides. ⁎ significantly different from control offspring of the same age.
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adipocytes were measured per animal (n = 5), and 250 adipocytes were measured per group. The areas were expressed in μm2.
Hepatic determination of triglyceride and glycogen After the animals were euthanized, 50 mg from the liver was homogenized in 1 mL isopropanol (Vetec, Rio de Janeiro, Brazil) and centrifuged (5900 rpm, 10 min, 4 °C). The total triglyceride level was measured using a colorimetric method with a commercial kit (Bioclin, Belo Horizonte, Brazil). The glucose produced through glycogen hydrolysis was measured using a commercial kit (Glucox, Doles, Goiânia, GO, Brazil). The liver was homogenized with 4 mL of TCA (10%) and then centrifuged (1000 g, 10 min, 4 °C). The supernatant (2 mL) was added to 5 mL of absolute ethanol and frozen. The mixture was centrifuged after 24 h (1000 g, 10 min, 4 °C), and the supernatant was discarded. The glycogen was hydrolyzed through boiling the pellet for 30 min with 1 M HCl. After adding 1 mL of 1 M NaOH to neutralize the mixture, glucose was measured in 200 μL of the supernatant (Casimiro-Lopes et al., 2012).
Assessment of redox balance (liver and plasma) To measure the antioxidant enzyme activities, liver samples (200 mg) were homogenized in potassium phosphate buffer with EDTA using a mechanical homogenizer (the CT-136 model from Cientec—laboratory equipment, Campinas, SP, Brazil). After centrifugation, the homogenates and plasma were stored at −80 °C until analysis and the total protein content was determined using the Bradford method (Bradford, 1976). The analyses were performed in accordance with a previous study (Conceição et al., 2013a,b). Briefly, the total superoxide dismutase (SOD) activity was assayed by measuring adrenaline auto-oxidation inhibition through the absorbance at 480 nm. The catalase (CAT) activity was measured as the rate of decrease in H2O2 at 240 nm. The glutathione peroxidase (GPx) activity was evaluated by measuring NADPH oxidation at 340 nm in the presence of H2O2. The radical nitric oxide (NO) was indirectly quantified by measuring nitrite (NO2−) in the liver at 540 nm. Additionally, lipid peroxidation was estimated by the malondialdehyde (MDA) concentration using the thiobarbituric acid reactive substance (TBARS) method. The protein oxidation was measured by reacting the carbonyl groups with 2,4-dinitrophenylhydrazine (Sigma-Aldrich, St. Louis, MO, USA). The absorbance values at 380 nm were measured and expressed as nmol of carbonyl per 0.5 mg of protein.
Table 3 Hepatic parameters of rats whose were early weaned at PN90 and PN 180. PN90
Weight liver (g/ mm) Range Glycogen (mM/g) Range TAG (mg/dL)/mg Range AST (U/L) Range ALT (U/L) Range
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Western blotting analysis To collect the cell extracts, 100 mg from the liver was homogenized in an ice-cold lysis buffer with protease inhibitors. After centrifugation, the homogenate protein concentration was determined using a specific kit (BCA Protein Assay Reagent, Thermo Scientific, IL, USA) and stored at − 20 °C. Briefly, the samples (10 mg from the total protein) were separated based on the molecular weight of each protein using 10% or 12% SDS-PAGE and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Amersham, London, UK). A standard rainbow marker (GE Healthcare, Little Chalfont, UK) was used in parallel to estimate the molecular weight. The membrane was blocked with 2% (w/v) BSA in T-TBS (0.02 M Tris pH 7.5, 0.15 M NaCl and 0.1% Tween 20) at room temperature for 1 h and washed with T-TBS. The membrane was then incubated with an anti-rabbit Cu–Zn SOD1 polyclonal antibody, anti-mouse MnSOD SOD2 monoclonal antibody, anti-mouse CAT monoclonal antibody, anti-mouse Gpx1/2 monoclonal antibody, and an anti-goat 4-HNE polyclonal antibody overnight at 4 °C. The primary antibodies were purchased from Santa Cruz Biotechnology, CA, USA and Sigma-Aldrich, St. Louis, MO, USA. After successive washes with T-TBS, the membrane was incubated with an appropriate secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h and then incubated with streptavidin (Zymed, San Francisco, CA, USA) at the same dilution as the secondary antibody for 1 h. The target proteins were detected with enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA) using the ImageQuant LAS 500 (GE Healthcare, Little Chalfont, UK). The images were quantified through densitometry using the Image J software (Media Cybernetics, Silver Spring, MD, USA). The results were normalized using a control. RT-PCR analysis The total RNA from the liver samples was extracted using the TRIzol reagent (Cat. No. 15596-018, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. We used 1 μg of the total RNA to prepare the cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Invitrogen, Carlsbad, CA, USA) and Oligo (dT) 15 Primer (Promega, Madison, WI, USA). The liver SIRT1 mRNA was amplified using the Applied Biosystems 7500 Real-Time PCR System (Life Technologies Co., Carlsbad, CA, USA) and the SYBR Green PCR Master Mix (Applied BioSystems, Foster City, CA, USA) in accordance the manufacturers' recommendations. The SIRT1 oligonucleotide primer sequences used were 5′-CAG GTT GCA GGA ATC CAA A-3′ (F) and 5′CAA ATC AGG CAA GAT GCT GT-3′ (R). The gene expression levels were normalized using 36-β4, an internal control, with the sequences 5′-TGT TTG ACA ACG GCA GCA TTT-3′ (F) and 5′-CCG AGG CAA CAG TTG GGT A-3′ (R). The product specificities were verified using a melting curve, and the assay was performed in triplicate using the 2ΔΔCT method. Statistical analysis
PN180
C
BRO
C
BRO
0.20 ± 0.01
0.20 ± 0.01
0.21 ± 0.01
0.22 ± 0.01
0.17–0.23 3.10 ± 0.89 1.12–6.34 1.56 ± 0.16 1.18–2.18 190.4 ± 29.34 88.0–264.0 75.60 ± 1.94 70.0–80.0
0.17–0.23 3.41 ± 0.38 2.45–4.36 1.72 ± 0.32 1.13–2.97 188.0 ± 16.25 139.0–213.0 67.40 ± 4.72 57.0–83.0
0.18–0.22 2.07 ± 0.69 0.22–4.02 1.68 ± 0.14 1.37–2.15 244.6 ± 18.66 191.0–308.0 75.40 ± 5.89 58.0–93.0
0.20–0.24 2.01 ± 0.19 1.59–2.69 2.54 ± 0.31⁎ 1.74–3.67 281.2 ± 9.83 256.0–306.0 83.40 ± 5.09 66.0–94.0
Values are mean and SEM. P b 0.05; n = 5 per group. C —control offspring; BRO —earlyweaned offspring; TAG —triglycerides; AST —aspartate aminotransferase; ALT —alanine aminotransferase. ⁎ significantly different from control offspring of the same age.
The data are the mean and standard error of mean (SEM). The differences between the groups were evaluated with Student's t test using GraphPad Prism 5 (Graph Pad Software, San Diego, California, USA). For each evaluation, P b 0.05 was considered to be statistically significant. Results Biometric data, adiposity and food intake Early weaning promotes a slight but significant BM reduction in the BRO offspring at the end of lactation (20 and 21 days old, − 6% and −8%, respectively, Table 1) without affecting the NAL. Thereafter, significant differences were not detected for both parameters during
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Fig. 2. Liver stereology. Photomicrographs of the liver with same magnification (×60) and stained with hematoxylin–eosin (HE) of C and BRO at PN90 and PN180, demonstrating a preservation of hepatic architecture in the BRO offspring. C —control offspring; BRO —early-weaned offspring.
the follow-up period and at the ages evaluated (PN90 and PN180, Table 1). The food intake did not differ between the groups for any time point. The increased adiposity in the BRO offspring was confirmed by measuring the VFM at PN90 (+ 45%) and PN180 (+ 47%), as shown in Table 1. Additionally, we observed adipocyte hypertrophy at PN90 (+ 31%) and PN180 (+ 43%), as demonstrated in the morphometric study (Fig. 1).
Metabolic profile The fasting glucose did not differ at PN90 and PN180. However, the OGTT area under curve increased (+ 10%) for the BRO offspring at PN90; altered glucose homeostasis is characteristic of glucose intolerance. In contrast, the BRO offspring OGTT did not differ at PN180 (Table 2). At PN90, the insulin and HOMA-IR remained unchanged for the BRO offspring. However, as expected, this group exhibited fasting hyperinsulinemia (+ 27%) and an increase in HOMA-IR (+ 30%) for PN180, which is characteristic of insulin resistance onset (Table 2). For the lipid profile, the BRO offspring showed differences in the plasma TAG (+52%) at PN180 (Table 2).
Liver analyses The liver weight/volume did not differ significantly between the groups at any time point studied. Higher triglyceride levels at PN180 (+ 51%) were only observed in the colorimetric assays. The other parameters analyzed (glycogen and hepatic enzymes) did not differ between the groups at both time points (Table 3). The liver photomicrographs were evaluated to identify marked tissue changes through ectopic fat deposition (steatosis), inflammatory infiltration, and stellate cell activation. However, no such morphological changes were detected between the groups evaluated at both time points (Fig. 2).
Redox status In the plasma assays, the BRO offspring exhibited higher SOD activity (+ 72%), higher MDA levels (+ 41%), higher GPx activity trends (P = 0.083), and higher protein carbonyl levels at PN90 (P = 0.077) (Fig. 3). At PN180, we detected lower SOD activity (− 25%), higher GPx activity (+ 38%), and lower MDA levels (− 88%) (Fig. 3). In the liver assays, the BRO offspring showed higher SOD activity at both time points (+ 88% at PN90 and + 72% at PN180), GPx activity at PN90 (+ 102%), and higher CAT activity at PN180 (+ 25%). This condition likely contributes to lower MDA levels at both time points (−30% at PN90 and −44% at PN180) (Fig. 3). The nitrite/nitrate content ratio did not differ at PN90 and PN180 (Fig. 3). The Western blot analyses for SOD-1, SOD-2, CAT, GPx, and 4-HNE did not differ between the groups at both time points (Fig. 4). The hepatic SIRT1 RT-PCR analyses showed a 4-fold increase in such expression for the BRO offspring compared with the C offspring at PN90 and a slight trend toward reduction at PN180 (Fig. 5). Discussion To determine whether early weaning is a risk factor for metabolic programming, certain experiments, such as maternal deprivation or mechanical breastfeeding interruption, have been designed to mimic this condition. However, both models imply a stressful event and complete lack of milk. The maternal bromocriptine treatment (Bonomo et al., 2005, 2007) inhibits prolactin and consequently reduces milk yield without full attenuation and maintains the maternal presence, which may be more similar to human weaning. However, one possible limitation for this model is that bromocriptine can be transferred through the milk to the pups, but we have shown that the serum prolactin levels do not change for the BRO offspring at PN21 (de Moura et al., 2009). However, bromocriptine may have a direct antioxidant action (Yoshikawa et al., 1994) and reduce both lipid peroxidation (Kline et al., 2004) and hyperphagia in adult obese rats, which improves the body composition and blood glucose (Davis et al., 2009).
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Fig. 3. Evaluation of antioxidant system and stress oxidative. (A) SOD activity in plasma and liver at PN90 and PN180; (B) GPx activity in plasma and liver at PN90 and PN180; (C) CAT activity in plasma and liver at PN90 and PN180; (D) MDA content in plasma and liver at PN90 and PN180; (E) total carbonyl protein content in plasma and liver at PN90 and PN180; (F) nitrite/nitrate content ratio in plasma and liver at PN90 and PN180. C —control offspring; BRO —early-weaned offspring; SOD —superoxide dismutase; GPx —glutathione peroxidase; CAT —catalase; MDA —malondialdehyde. Values are means and SEM. P b 0.05. n = 5 per group.
Additionally, bromocriptine (10 mg/kg over 4 weeks) was effective at treating liver steatosis in genetically modified obese rats (Zucker rats) (Davis et al., 2006). These authors speculated that the bromocriptine antioxidants effects (increased MnSOD, as demonstrated with immunohistochemistry) may have caused the improved hepatic histology. In another study, the authors tested several bromocriptine doses in rats under alcohol-induced oxidative stress, and the group treated with the minor dose (12.5 mg/kg) yielded better liver parameters related to antioxidant system (Popovic et al., 2008). Thus, using bromocriptine in the pharmacological early weaning model may be a confounding factor in milk inhibition because it may regulate redox status, adipogenesis, and glucose homeostasis alone, given that this drug yields antioxidant, anti-obesity, and anti-diabetogenic effects. Therefore, the data presented show a superior redox status and relative protection from liver dysfunction despite the metabolic disorders and obesity in the adult
progeny, suggesting that bromocriptine may have a specific imprinting role that was not observed in other early weaning models. We corroborate a previous study, wherein this programming model had increased visceral adiposity (de Moura et al., 2009), and the histological analyses that demonstrate adipocyte hypertrophy at PN90 and PN180 provide further validation. Thus, the effects of undernutrition during the critical lactation period outweigh the possible beneficial effects from bromocriptine on body composition. This fat deposit hypertrophy may be a key pathophysiological link between obesity and diabetes onset through the adipokines secretion deregulation and increased release of free fatty acids that contribute to insulin resistance (Dunmore and Brown, 2013). Thus, the BRO offspring fasting glucose, OGTT and plasma insulin suggest a pre-diabetic status, for which hyperinsulinemia apparently compensated (normal AUC glucose) by PN180; however, the HOMA-IR results suggest insulin resistance.
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Fig. 4. Western blotting in liver tissue. (A) Protein content of Cu/Zn SOD; (B) catalase; (C) 4-HNE; (D) MnSOD; (E) GPx. C —control offspring; BRO —early-weaned offspring; SOD —superoxide dismutase; GPx —glutathione peroxidase; 4-HNE —4-hydroxy-2-nonenal; SIRT1 —sirtuin 1. Values are means and SEM. P b 0.05. n = 5 per group.
events. Thus, altered adiposity and glucose homeostasis increases oxidative stress, which triggers a greater adaptive antioxidant response that corrects the lipid peroxidation, particularly in the liver. Herein, we evaluated such responses not only using the TBARS method, which may have certain limitations, but also 4-HNE.
3
RNAm SIRT1/36 β
Data on the redox balance for the BRO offspring suggest that a process that minimized oxidative stress was triggered by PN90; this process was characterized by a higher plasma MDA and may have become more effective by PN180 because the plasma MDA was markedly reduced. In younger animals, altered glucose homeostasis promotes greater reactive oxygen species production (Kassab and Piwowar, 2012), which can cause a compensatory increase in SOD activity and an increasing trend in GPx activity to minimize MDA and protein carbonyl formation. In older animals, the increased GPx activity compensates for the reduced SOD activity, which remains reduced during oxidative stress. Paradoxically, the lower SOD activity can contribute to lower lipid peroxidation levels because it is associated with reduced hydrogen peroxide formation from the reduced SOD-mediated superoxide anion dismutation (Kar et al., 2012). Using the same experimental design, a previous study from our laboratory reported an increase in oxidative stress in PN90 BRO offspring plasma followed by a decrease in the total antioxidant capacity, which was analyzed by DPPH reduction. This condition was apparently improved by PN180 (Casimiro-Lopes et al., 2012). Herein, to understand the mechanism involved in the improved redox status, we evaluated the antioxidant system by studying other oxidative stress biomarkers and by evaluating the hepatic oxidative stress and antioxidant system for the first time, which allowed us to better describe the sequence of
*
2
1
0 P90
P180
Fig. 5. RT-PCR in liver tissue at PN90 and PN180. Protein expression —mRNA SIRT1. C —control offspring; BRO —early-weaned offspring; SIRT1 —sirtuin 1. Values are means and SEM. P b 0.05. n = 5 per group.
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In the liver morphological evaluations, it is interesting that the BRO offspring did not present alterations by PN180, despite the higher hepatic triglyceride levels; at this time point, the enhanced lipids did not produce hepatic morphological alterations. This result differs from the non-pharmacological early weaning model, wherein the dams were wrapped with bandages to interrupt lactation for the same time period studied in the current study (Franco et al., 2013). In this model, the offspring were programmed for liver microsteatosis and enhanced plasma and hepatic MDA levels, which are associated with a diminished antioxidant capacity (i.e., plasma SOD activity and liver GPx activity) during adulthood. Thus, the current study shows an improved redox status, wherein MDA is lower in both the plasma and liver primarily because of enhanced plasma and hepatic GPx and liver SOD activities, despite a similar decrease in plasma SOD activity. Curiously, in the non-pharmacological early weaning model, in which the redox status was worse during adulthood, the redox status was fully reversed by resveratrol treatment (Franco et al., 2013). These observations may have been because of an increase in SIRT1, which can inhibit important pathways for nonalcoholic fatty liver disease (NAFLD) pathogenesis and oxidative stress (Kaeberlein et al., 2005). We observed higher hepatic mRNA SIRT1 expression in the PN90 BRO offspring; this result may have caused the enhanced antioxidant capacity that aided in the liver tissue protection because early weaning can impair liver function. We suggest that SIRT1 may increase before the PN90 time point; however, additional experiments must be performed to confirm this hypothesis. To the best of our knowledge, previous studies have not reported on the effect of bromocriptine on SIRT1 activity or expression. Although liver function is preserved at PN180, SIRT1 does not continue to increase by this time point. Thus, the reduced SIRT1 expression helps explain certain unfavorable alterations that were observed in the PN180 BRO group, such as insulin resistance, higher liver triglycerides, and lower plasmic SOD activity; these activities may have also been observed if the study had been extended to later time points. Conclusion The long-term effects from programming by early weaning were likely induced through neonatal undernutrition and, as expected, produced metabolic disruptions that are characteristic of obesity. However, the study results suggest that bromocriptine programming improved the redox status by protecting against nonalcoholic fatty liver disease, perhaps through SIRT1. The observations in this study further encourage additional studies to better understand the effects of bromocriptine during this critical life stage and its potential therapeutic use in preventing developmental diseases. Conflict of interest statement The authors have no financial and commercial conflicts of interest.
Acknowledgments All authors are grateful to Miss Monica Moura and Mr. Ulisses Risso Siqueira for technical assistance. This research was supported by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico — CNPq), the Coordination for the Enhancement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior —CAPES) and the State of Rio de Janeiro Carlos Chagas Filho Research Foundation (Fundacão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro —FAPERJ).
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Does bromocriptine play a role in decreasing oxidative stress for early weaned programmed obesity? Nayara Peixoto-Silva a, Ellen P.S. Conceição a, Janaine C. Carvalho a, Natália S. Lima a, José Firmino Nogueira-Neto b, Elaine de Oliveira a, Egberto G. Moura a, Patricia C. Lisboa a,⁎ a b
Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, Brazil Laboratory of Lipids, School of Medicine, State University of Rio de Janeiro, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 1 August 2013 Accepted 6 December 2013 Keywords: Early weaning Bromocriptine Obesity Liver disease Antioxidant system
a b s t r a c t Aims: Studies have demonstrated that early weaning can promote metabolic syndrome during adulthood and that obesity increases oxidative stress. Thus, we aimed to evaluate redox status in a pharmacological early weaning rodent model programmed for metabolic syndrome at adulthood. Main methods: Lactating dams were randomly assigned into 2 groups: the early weaning group (BRO), which was treated intraperitoneally with bromocriptine (1 mg/day) to inhibit prolactin secretion for the last 3 days of lactation, and the control group (C), which received the BRO diluent for the same time period. The offspring were killed at 90 (PN90) and 180 (PN180) days after birth. Key findings: Early weaning induced greater visceral adiposity and dyslipidemia. At PN90, the BRO offspring showed glucose intolerance with normoinsulinemia and increased plasma and liver superoxide dismutase, and liver glutathione peroxidase activities, which reduced the liver malondialdehyde but not the increased plasma malondialdehyde levels. However, the BRO offspring showed insulin resistance at PN180 and increased plasma glutathione peroxidase, liver superoxide dismutase, and catalase activities. These changes reduced the plasma and liver malondialdehyde levels, which aided in hepatocyte architecture preservation. Additionally, we observed that sirtuin 1 was overexpressed in the BRO group at PN90, but the increased expression was not maintained through PN180, which suggests unfavorable metabolic conditions in the older offspring. Significance: Despite the observed obesity and glucose homeostasis dysfunction, our data suggest that the early weaning programming induced by bromocriptine can improve the offspring's redox status and may prevent liver damage during adulthood. © 2013 Elsevier Inc. All rights reserved.
Introduction Breastfeeding is associated with reduced obesity risk because maternal milk causes hypoinsulinemia, which decreases fat storage and prevents excessive early adipogenesis (Oddy, 2012). In fact, early weaning is related to childhood obesity, which demonstrates that the timing for introducing solid food to an infant's diet affects weight gain (Sloan et al., 2008). Studies were performed to understand the mechanisms involved in the later effects triggered by early weaning. When pups were weaned through maternal separation, the offspring showed a preference for high-calorie food during adulthood, which led to obesity and metabolic changes (dos Santos et al., 2011). Additional alterations were observed during adulthood after early weaning from lactation by applying a ⁎ Corresponding author at: Departamento de Ciências Fisiológicas, 5° andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ, 20551–030, Brazil. Tel.: +55 21 28688334; fax: +55 21 28688029. E-mail address: [email protected] (P.C. Lisboa). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.013
bandage on the mother's body to interrupt lactation without maternal separation. The adult offspring exhibited obesity, dyslipidemia, and hyperglycemia, which are characteristics of metabolic syndrome, hyperleptinemia, central leptin resistance, and higher catecholamine (Lima Nda et al., 2011; Lima et al., 2013). Further, milk production can be suppressed in a pharmacological model for early weaning using bromocriptine, which is a type 2 dopaminergic receptor agonist that inhibits prolactin. Offspring with mothers treated with bromocriptine exhibited neonatal malnutrition and developed obesity, hyperleptinemia, leptin and insulin resistance, dyslipidemia, hypothyroidism, and higher adrenal hormones during adulthood (Bonomo et al., 2007, 2008; de Moura et al., 2009). Long-lasting dysfunction induced by early weaning may lead to liver changes; the liver is a central metabolic organ that regulates energy homeostasis. In nonalcoholic fatty liver disease, metabolic syndrome is manifested; in insulin resistance, oxidative stress and inflammatory cascades can be important for hepatic disease pathogenesis and progression (Colak et al., 2011). Thus, oxidative stress is an important mechanism that links obesity and its comorbidities because both a
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suppressed antioxidant system and an enhanced reactive oxygen species production are related to central adiposity in obese individuals (Savini et al., 2013). Recently, a non-pharmacological early weaning model was programmed for increased oxidative stress and steatosis in the liver, which was reversed through resveratrol treatment. Resveratrol is a natural phytoalexin in grapes, which produces beneficial effects because it is an antioxidant agent (Franco et al., 2013) that activates histone deacetylases, sirtuin 1 (SIRT1), and aids in treating or preventing obesity. Notably, (i) lactation is critical for development, and (ii) a pharmacological early weaning model induces several disruptions in adult progeny that are similar to the disruptions observed in a non-pharmacological early weaning model, such as metabolic syndrome. Therefore, the study herein was designed to evaluate glucose homeostasis, redox status, and hepatic morphological alterations at 2 different time points in adult offspring whose mothers received bromocriptine at the end of lactation. For insight into the programming mechanisms, liver SIRT1 expression was measured in our study. SIRT1 is important for glucose and lipid metabolism (Guarente, 2006), and higher SIRT1 activity has been related to lower adiposity and protection against diet-induced metabolic disorders (Pfluger et al., 2008); therefore, we reasoned that SIRT1 expression may be decreased by early weaning.
Material and methods Ethical approval The experiment was designed and performed in accordance with the principles adopted and promulgated by Brazilian law no. 11.794/2008, and each procedure was approved by the Animal Care and Use Committee at the Biology Institute in the State University of Rio de Janeiro (CEUA/186/2007, CEUA/017/2009). The experiment was performed to minimize the number of animals used and the suffering from the procedures following the three ‘R’s' of ethical doctrine: reduction, refinement, and replacement (Drummond, 2009).
Animals The animals were maintained in a temperature-controlled room (23/24 °C) with artificial dark–light cycles (lights on for 7 h and lights off for 19 h). Three-month-old virgin female Wistar rats were mated with male breeders at a 2:1 ratio. The pregnant rats were maintained in individual cages with free access to water and standard chow until delivery and during lactation. To avoid the influence of litter size on the programming effect, only the mothers with 10 pups were used. After spontaneous delivery, the litters were adjusted to 6 male pups by the mother (anogenital distance was used to determine the gender). Manipulating the litter standardized the food supply and maximized the lactation performance. Beginning at birth, the body mass (BM) and naso-anal length (NAL) were measured in the male pups. The lactating dams were randomly assigned to 2 groups: (BRO, n = 5), which was administered 2 doses of 0.5 mg bromocriptine (Novartis, SP, Brazil) per day (8:00 AM and 8:00 PM) diluted in 200 μL of methanol–saline (1:1 v/v) and injected intraperitoneally for the last 3 days of lactation, and the control group (C, n = 5), which received only a methanol–saline treatment for the same time period. After weaning, 1 pup was randomly selected from each litter (5 animals per group) and analyzed 90 days after birth (PN90). The analyses at 180 days after birth (PN180) were performed using the average between 2 randomly selected pups from each litter, also totaling 5 animals per group.
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Biometric parameters and food intake During lactation, the BM and NAL were measured every 3 days; after weaning, the absolute values for the parameters were measured weekly with the food intake. The food consumption was measured as the difference between the amount of food provided and remaining 7 days later divided by the number of animals in the cage. Oral glucose tolerance test An oral glucose tolerance test (OGTT) was performed at PN90 and PN180. After a 12 hour fasting period, 50% glucose was administered in sterile saline (0.9% NaCl) through an oral gavage at 2 g/kg BM. Blood was drawn from the tip tail of each animal to measure the plasma glucose concentration, which was assessed using a glucometer (Accu-Chek Advantage; Roche Diagnostics, Mannheim, Germany) before the glucose was administered and 15, 30, 60, and 120 min after the gavage (Peixoto-Silva et al., 2011). Euthanasia After fasting for 12 h, the adult rats were sacrificed by quick decapitation to collect the blood, visceral fat mass ([VFM], retroperitoneal, epididymal, and mesenteric fat) and liver. The blood was previously collected in a heparinized tube and centrifuged (2500 rpm, 25 min, 4 °C). The plasma was collected and stored individually at −20 ° C until it was used for the different measurements. The hydrostatic liver weight/ volume was measured using the Scherle method and normalized to the right tibia length in accordance with previous studies (Conceição et al., 2013a,b). The tissues were fractionated and stored using multiple procedures (freezing at −80 °C or fixative solution). Plasma insulin and biochemical analyses The insulin concentration was determined using an RIA kit (ICN Pharmaceuticals, Inc., Orangeburg, NY, USA) with the assay sensitivity 0.1 ng/mL and a 4.1% intra-assay variation. The measurements were performed using a single assay. To determine the adult animals' insulin sensitivity, we used the homeostatic model assessment of insulin resistance (HOMA-IR): (fasting glycemia [mmol/L] × fasting insulinemia [μU/mL] / 22.5). We analyzed the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglyceride (TAG) plasma levels using Biosystem commercial test kits and a spectrophotometer (Biosystems S.A., Barcelona, Spain). Histological processing and adipocyte morphometry Fragments from the retroperitoneal fat and liver were collected from each lobe and fixed in a freshly prepared fixative (1.27 M—formaldehyde, Table 1 Biometry and adiposity of rats whose were early weaned. C BM (g) Birth 18 days old 19 days old 20 days old 21 days old PN90 PN180 VFM (g) PN90 (g) PN180 (g)
6.125 38.43 41.07 43.89 46.62 323.8 398.2
BRO ± ± ± ± ± ± ±
0.08 0.61 0.69 0.62 0.61 9.08 14.03
7.82 ± 0.33 9.44 ± 0.96
5.955 38.05 39.91 41.52 42.97 331.6 421.0
± ± ± ± ± ± ±
0.09 0.79 0.79 0.87⁎ 0.98⁎ 20.24 15.99
11.40 ± 1.51⁎ 13.87 ± 1.64⁎
Values are mean and SEM. P b 0.05; n = 5 per group. C —control offspring; BRO —earlyweaned offspring; BM —body mass; VFM —visceral fat mass. ⁎ significantly different from control offspring.
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Fig. 1. Mean cross-sectional area of adipocytes. Photomicrographs of the retroperitoneal fat with same magnification (×40) and stained with hematoxylin–eosin (HE) demonstrating a hypertrophy of adipocytes in the BRO offspring at PN90 and PN180. C —control offspring; BRO —early-weaned offspring. Values are means and SEM. P b 0.05. n = 5 per group.
0.1 M—phosphate buffer, pH 7.2) at room temperature for 48 h. After histological processing, the tissues were embedded in Paraplast Plus ® (Sigma-Aldrich, St. Louis, MO, USA) to produce random 5 μm thick cuts and stained with hematoxylin–eosin (HE) for visualization using light microscopy.
The adipocyte sectional areas were analyzed using Image-Pro Plus software, version 5.0 (Media Cybernetics, Silver Spring, MD, USA) with randomly acquired digital images (TIFF format, 36-bit color, 1360 × 1024 pixels, 40 ×) using an Olympus BX40 microscope with an Olympus DP71 and camera (Olympus, Tokyo, Japan). In total, 50
Table 2 Metabolic profile of rats whose were early weaned at PN90 and PN180. PN90
Fasting glucose (mg/dL) Range OGTT (AUC) Range Serum insulin (μUI/mL) Range HOMA-IR Range TAG (mg/dL) Range
PN180
C
BRO
C
BRO
70 ± 1.59 64.0–73.0 12,200 ± 267.1 11,408–12,990 31.01 ± 1.87 25.05–36.8 5.38 ± 0.32 4.263–6127 51.0 ± 3.35 42.0–58.0
69 ± 2.65 61.0–77.0 13,460 ± 323.5⁎ 12,180–13,950 35.24 ± 2.66 27.01–43.11 6.01 ± 0.61 4731–8189 61.8 ± 3.97 55.0–76.0
74 ± 4.92 64.5–92.0 14,020 ± 338.0 11,468–15,210 35.79 ± 2.97 25.95–43.04 5.94 ± 0.46 4.75−7.48 55.8 ± 2.35 47.0–60.0
68 ± 2.11 60.5–72.0 14,000 ± 373.6 12,398–17,055 45.57 ± 2.43⁎ 40.71–54.43 7.77 ± 0.56⁎ 6.21–9.50 85.1 ± 10.79⁎ 50.0–113.0
Values are mean and SEM. P b 0.05; n = 5 per group. C —control offspring; BRO —early-weaned offspring; OGTT —oral glucose tolerance test; AUC —area under curve; HOMA-IR —homeostatic model assessment of insulin resistance; TAG— triglycerides. ⁎ significantly different from control offspring of the same age.
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adipocytes were measured per animal (n = 5), and 250 adipocytes were measured per group. The areas were expressed in μm2.
Hepatic determination of triglyceride and glycogen After the animals were euthanized, 50 mg from the liver was homogenized in 1 mL isopropanol (Vetec, Rio de Janeiro, Brazil) and centrifuged (5900 rpm, 10 min, 4 °C). The total triglyceride level was measured using a colorimetric method with a commercial kit (Bioclin, Belo Horizonte, Brazil). The glucose produced through glycogen hydrolysis was measured using a commercial kit (Glucox, Doles, Goiânia, GO, Brazil). The liver was homogenized with 4 mL of TCA (10%) and then centrifuged (1000 g, 10 min, 4 °C). The supernatant (2 mL) was added to 5 mL of absolute ethanol and frozen. The mixture was centrifuged after 24 h (1000 g, 10 min, 4 °C), and the supernatant was discarded. The glycogen was hydrolyzed through boiling the pellet for 30 min with 1 M HCl. After adding 1 mL of 1 M NaOH to neutralize the mixture, glucose was measured in 200 μL of the supernatant (Casimiro-Lopes et al., 2012).
Assessment of redox balance (liver and plasma) To measure the antioxidant enzyme activities, liver samples (200 mg) were homogenized in potassium phosphate buffer with EDTA using a mechanical homogenizer (the CT-136 model from Cientec—laboratory equipment, Campinas, SP, Brazil). After centrifugation, the homogenates and plasma were stored at −80 °C until analysis and the total protein content was determined using the Bradford method (Bradford, 1976). The analyses were performed in accordance with a previous study (Conceição et al., 2013a,b). Briefly, the total superoxide dismutase (SOD) activity was assayed by measuring adrenaline auto-oxidation inhibition through the absorbance at 480 nm. The catalase (CAT) activity was measured as the rate of decrease in H2O2 at 240 nm. The glutathione peroxidase (GPx) activity was evaluated by measuring NADPH oxidation at 340 nm in the presence of H2O2. The radical nitric oxide (NO) was indirectly quantified by measuring nitrite (NO2−) in the liver at 540 nm. Additionally, lipid peroxidation was estimated by the malondialdehyde (MDA) concentration using the thiobarbituric acid reactive substance (TBARS) method. The protein oxidation was measured by reacting the carbonyl groups with 2,4-dinitrophenylhydrazine (Sigma-Aldrich, St. Louis, MO, USA). The absorbance values at 380 nm were measured and expressed as nmol of carbonyl per 0.5 mg of protein.
Table 3 Hepatic parameters of rats whose were early weaned at PN90 and PN 180. PN90
Weight liver (g/ mm) Range Glycogen (mM/g) Range TAG (mg/dL)/mg Range AST (U/L) Range ALT (U/L) Range
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Western blotting analysis To collect the cell extracts, 100 mg from the liver was homogenized in an ice-cold lysis buffer with protease inhibitors. After centrifugation, the homogenate protein concentration was determined using a specific kit (BCA Protein Assay Reagent, Thermo Scientific, IL, USA) and stored at − 20 °C. Briefly, the samples (10 mg from the total protein) were separated based on the molecular weight of each protein using 10% or 12% SDS-PAGE and transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech, Amersham, London, UK). A standard rainbow marker (GE Healthcare, Little Chalfont, UK) was used in parallel to estimate the molecular weight. The membrane was blocked with 2% (w/v) BSA in T-TBS (0.02 M Tris pH 7.5, 0.15 M NaCl and 0.1% Tween 20) at room temperature for 1 h and washed with T-TBS. The membrane was then incubated with an anti-rabbit Cu–Zn SOD1 polyclonal antibody, anti-mouse MnSOD SOD2 monoclonal antibody, anti-mouse CAT monoclonal antibody, anti-mouse Gpx1/2 monoclonal antibody, and an anti-goat 4-HNE polyclonal antibody overnight at 4 °C. The primary antibodies were purchased from Santa Cruz Biotechnology, CA, USA and Sigma-Aldrich, St. Louis, MO, USA. After successive washes with T-TBS, the membrane was incubated with an appropriate secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h and then incubated with streptavidin (Zymed, San Francisco, CA, USA) at the same dilution as the secondary antibody for 1 h. The target proteins were detected with enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA) using the ImageQuant LAS 500 (GE Healthcare, Little Chalfont, UK). The images were quantified through densitometry using the Image J software (Media Cybernetics, Silver Spring, MD, USA). The results were normalized using a control. RT-PCR analysis The total RNA from the liver samples was extracted using the TRIzol reagent (Cat. No. 15596-018, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. We used 1 μg of the total RNA to prepare the cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Invitrogen, Carlsbad, CA, USA) and Oligo (dT) 15 Primer (Promega, Madison, WI, USA). The liver SIRT1 mRNA was amplified using the Applied Biosystems 7500 Real-Time PCR System (Life Technologies Co., Carlsbad, CA, USA) and the SYBR Green PCR Master Mix (Applied BioSystems, Foster City, CA, USA) in accordance the manufacturers' recommendations. The SIRT1 oligonucleotide primer sequences used were 5′-CAG GTT GCA GGA ATC CAA A-3′ (F) and 5′CAA ATC AGG CAA GAT GCT GT-3′ (R). The gene expression levels were normalized using 36-β4, an internal control, with the sequences 5′-TGT TTG ACA ACG GCA GCA TTT-3′ (F) and 5′-CCG AGG CAA CAG TTG GGT A-3′ (R). The product specificities were verified using a melting curve, and the assay was performed in triplicate using the 2ΔΔCT method. Statistical analysis
PN180
C
BRO
C
BRO
0.20 ± 0.01
0.20 ± 0.01
0.21 ± 0.01
0.22 ± 0.01
0.17–0.23 3.10 ± 0.89 1.12–6.34 1.56 ± 0.16 1.18–2.18 190.4 ± 29.34 88.0–264.0 75.60 ± 1.94 70.0–80.0
0.17–0.23 3.41 ± 0.38 2.45–4.36 1.72 ± 0.32 1.13–2.97 188.0 ± 16.25 139.0–213.0 67.40 ± 4.72 57.0–83.0
0.18–0.22 2.07 ± 0.69 0.22–4.02 1.68 ± 0.14 1.37–2.15 244.6 ± 18.66 191.0–308.0 75.40 ± 5.89 58.0–93.0
0.20–0.24 2.01 ± 0.19 1.59–2.69 2.54 ± 0.31⁎ 1.74–3.67 281.2 ± 9.83 256.0–306.0 83.40 ± 5.09 66.0–94.0
Values are mean and SEM. P b 0.05; n = 5 per group. C —control offspring; BRO —earlyweaned offspring; TAG —triglycerides; AST —aspartate aminotransferase; ALT —alanine aminotransferase. ⁎ significantly different from control offspring of the same age.
The data are the mean and standard error of mean (SEM). The differences between the groups were evaluated with Student's t test using GraphPad Prism 5 (Graph Pad Software, San Diego, California, USA). For each evaluation, P b 0.05 was considered to be statistically significant. Results Biometric data, adiposity and food intake Early weaning promotes a slight but significant BM reduction in the BRO offspring at the end of lactation (20 and 21 days old, − 6% and −8%, respectively, Table 1) without affecting the NAL. Thereafter, significant differences were not detected for both parameters during
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Fig. 2. Liver stereology. Photomicrographs of the liver with same magnification (×60) and stained with hematoxylin–eosin (HE) of C and BRO at PN90 and PN180, demonstrating a preservation of hepatic architecture in the BRO offspring. C —control offspring; BRO —early-weaned offspring.
the follow-up period and at the ages evaluated (PN90 and PN180, Table 1). The food intake did not differ between the groups for any time point. The increased adiposity in the BRO offspring was confirmed by measuring the VFM at PN90 (+ 45%) and PN180 (+ 47%), as shown in Table 1. Additionally, we observed adipocyte hypertrophy at PN90 (+ 31%) and PN180 (+ 43%), as demonstrated in the morphometric study (Fig. 1).
Metabolic profile The fasting glucose did not differ at PN90 and PN180. However, the OGTT area under curve increased (+ 10%) for the BRO offspring at PN90; altered glucose homeostasis is characteristic of glucose intolerance. In contrast, the BRO offspring OGTT did not differ at PN180 (Table 2). At PN90, the insulin and HOMA-IR remained unchanged for the BRO offspring. However, as expected, this group exhibited fasting hyperinsulinemia (+ 27%) and an increase in HOMA-IR (+ 30%) for PN180, which is characteristic of insulin resistance onset (Table 2). For the lipid profile, the BRO offspring showed differences in the plasma TAG (+52%) at PN180 (Table 2).
Liver analyses The liver weight/volume did not differ significantly between the groups at any time point studied. Higher triglyceride levels at PN180 (+ 51%) were only observed in the colorimetric assays. The other parameters analyzed (glycogen and hepatic enzymes) did not differ between the groups at both time points (Table 3). The liver photomicrographs were evaluated to identify marked tissue changes through ectopic fat deposition (steatosis), inflammatory infiltration, and stellate cell activation. However, no such morphological changes were detected between the groups evaluated at both time points (Fig. 2).
Redox status In the plasma assays, the BRO offspring exhibited higher SOD activity (+ 72%), higher MDA levels (+ 41%), higher GPx activity trends (P = 0.083), and higher protein carbonyl levels at PN90 (P = 0.077) (Fig. 3). At PN180, we detected lower SOD activity (− 25%), higher GPx activity (+ 38%), and lower MDA levels (− 88%) (Fig. 3). In the liver assays, the BRO offspring showed higher SOD activity at both time points (+ 88% at PN90 and + 72% at PN180), GPx activity at PN90 (+ 102%), and higher CAT activity at PN180 (+ 25%). This condition likely contributes to lower MDA levels at both time points (−30% at PN90 and −44% at PN180) (Fig. 3). The nitrite/nitrate content ratio did not differ at PN90 and PN180 (Fig. 3). The Western blot analyses for SOD-1, SOD-2, CAT, GPx, and 4-HNE did not differ between the groups at both time points (Fig. 4). The hepatic SIRT1 RT-PCR analyses showed a 4-fold increase in such expression for the BRO offspring compared with the C offspring at PN90 and a slight trend toward reduction at PN180 (Fig. 5). Discussion To determine whether early weaning is a risk factor for metabolic programming, certain experiments, such as maternal deprivation or mechanical breastfeeding interruption, have been designed to mimic this condition. However, both models imply a stressful event and complete lack of milk. The maternal bromocriptine treatment (Bonomo et al., 2005, 2007) inhibits prolactin and consequently reduces milk yield without full attenuation and maintains the maternal presence, which may be more similar to human weaning. However, one possible limitation for this model is that bromocriptine can be transferred through the milk to the pups, but we have shown that the serum prolactin levels do not change for the BRO offspring at PN21 (de Moura et al., 2009). However, bromocriptine may have a direct antioxidant action (Yoshikawa et al., 1994) and reduce both lipid peroxidation (Kline et al., 2004) and hyperphagia in adult obese rats, which improves the body composition and blood glucose (Davis et al., 2009).
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Fig. 3. Evaluation of antioxidant system and stress oxidative. (A) SOD activity in plasma and liver at PN90 and PN180; (B) GPx activity in plasma and liver at PN90 and PN180; (C) CAT activity in plasma and liver at PN90 and PN180; (D) MDA content in plasma and liver at PN90 and PN180; (E) total carbonyl protein content in plasma and liver at PN90 and PN180; (F) nitrite/nitrate content ratio in plasma and liver at PN90 and PN180. C —control offspring; BRO —early-weaned offspring; SOD —superoxide dismutase; GPx —glutathione peroxidase; CAT —catalase; MDA —malondialdehyde. Values are means and SEM. P b 0.05. n = 5 per group.
Additionally, bromocriptine (10 mg/kg over 4 weeks) was effective at treating liver steatosis in genetically modified obese rats (Zucker rats) (Davis et al., 2006). These authors speculated that the bromocriptine antioxidants effects (increased MnSOD, as demonstrated with immunohistochemistry) may have caused the improved hepatic histology. In another study, the authors tested several bromocriptine doses in rats under alcohol-induced oxidative stress, and the group treated with the minor dose (12.5 mg/kg) yielded better liver parameters related to antioxidant system (Popovic et al., 2008). Thus, using bromocriptine in the pharmacological early weaning model may be a confounding factor in milk inhibition because it may regulate redox status, adipogenesis, and glucose homeostasis alone, given that this drug yields antioxidant, anti-obesity, and anti-diabetogenic effects. Therefore, the data presented show a superior redox status and relative protection from liver dysfunction despite the metabolic disorders and obesity in the adult
progeny, suggesting that bromocriptine may have a specific imprinting role that was not observed in other early weaning models. We corroborate a previous study, wherein this programming model had increased visceral adiposity (de Moura et al., 2009), and the histological analyses that demonstrate adipocyte hypertrophy at PN90 and PN180 provide further validation. Thus, the effects of undernutrition during the critical lactation period outweigh the possible beneficial effects from bromocriptine on body composition. This fat deposit hypertrophy may be a key pathophysiological link between obesity and diabetes onset through the adipokines secretion deregulation and increased release of free fatty acids that contribute to insulin resistance (Dunmore and Brown, 2013). Thus, the BRO offspring fasting glucose, OGTT and plasma insulin suggest a pre-diabetic status, for which hyperinsulinemia apparently compensated (normal AUC glucose) by PN180; however, the HOMA-IR results suggest insulin resistance.
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Fig. 4. Western blotting in liver tissue. (A) Protein content of Cu/Zn SOD; (B) catalase; (C) 4-HNE; (D) MnSOD; (E) GPx. C —control offspring; BRO —early-weaned offspring; SOD —superoxide dismutase; GPx —glutathione peroxidase; 4-HNE —4-hydroxy-2-nonenal; SIRT1 —sirtuin 1. Values are means and SEM. P b 0.05. n = 5 per group.
events. Thus, altered adiposity and glucose homeostasis increases oxidative stress, which triggers a greater adaptive antioxidant response that corrects the lipid peroxidation, particularly in the liver. Herein, we evaluated such responses not only using the TBARS method, which may have certain limitations, but also 4-HNE.
3
RNAm SIRT1/36 β
Data on the redox balance for the BRO offspring suggest that a process that minimized oxidative stress was triggered by PN90; this process was characterized by a higher plasma MDA and may have become more effective by PN180 because the plasma MDA was markedly reduced. In younger animals, altered glucose homeostasis promotes greater reactive oxygen species production (Kassab and Piwowar, 2012), which can cause a compensatory increase in SOD activity and an increasing trend in GPx activity to minimize MDA and protein carbonyl formation. In older animals, the increased GPx activity compensates for the reduced SOD activity, which remains reduced during oxidative stress. Paradoxically, the lower SOD activity can contribute to lower lipid peroxidation levels because it is associated with reduced hydrogen peroxide formation from the reduced SOD-mediated superoxide anion dismutation (Kar et al., 2012). Using the same experimental design, a previous study from our laboratory reported an increase in oxidative stress in PN90 BRO offspring plasma followed by a decrease in the total antioxidant capacity, which was analyzed by DPPH reduction. This condition was apparently improved by PN180 (Casimiro-Lopes et al., 2012). Herein, to understand the mechanism involved in the improved redox status, we evaluated the antioxidant system by studying other oxidative stress biomarkers and by evaluating the hepatic oxidative stress and antioxidant system for the first time, which allowed us to better describe the sequence of
*
2
1
0 P90
P180
Fig. 5. RT-PCR in liver tissue at PN90 and PN180. Protein expression —mRNA SIRT1. C —control offspring; BRO —early-weaned offspring; SIRT1 —sirtuin 1. Values are means and SEM. P b 0.05. n = 5 per group.
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In the liver morphological evaluations, it is interesting that the BRO offspring did not present alterations by PN180, despite the higher hepatic triglyceride levels; at this time point, the enhanced lipids did not produce hepatic morphological alterations. This result differs from the non-pharmacological early weaning model, wherein the dams were wrapped with bandages to interrupt lactation for the same time period studied in the current study (Franco et al., 2013). In this model, the offspring were programmed for liver microsteatosis and enhanced plasma and hepatic MDA levels, which are associated with a diminished antioxidant capacity (i.e., plasma SOD activity and liver GPx activity) during adulthood. Thus, the current study shows an improved redox status, wherein MDA is lower in both the plasma and liver primarily because of enhanced plasma and hepatic GPx and liver SOD activities, despite a similar decrease in plasma SOD activity. Curiously, in the non-pharmacological early weaning model, in which the redox status was worse during adulthood, the redox status was fully reversed by resveratrol treatment (Franco et al., 2013). These observations may have been because of an increase in SIRT1, which can inhibit important pathways for nonalcoholic fatty liver disease (NAFLD) pathogenesis and oxidative stress (Kaeberlein et al., 2005). We observed higher hepatic mRNA SIRT1 expression in the PN90 BRO offspring; this result may have caused the enhanced antioxidant capacity that aided in the liver tissue protection because early weaning can impair liver function. We suggest that SIRT1 may increase before the PN90 time point; however, additional experiments must be performed to confirm this hypothesis. To the best of our knowledge, previous studies have not reported on the effect of bromocriptine on SIRT1 activity or expression. Although liver function is preserved at PN180, SIRT1 does not continue to increase by this time point. Thus, the reduced SIRT1 expression helps explain certain unfavorable alterations that were observed in the PN180 BRO group, such as insulin resistance, higher liver triglycerides, and lower plasmic SOD activity; these activities may have also been observed if the study had been extended to later time points. Conclusion The long-term effects from programming by early weaning were likely induced through neonatal undernutrition and, as expected, produced metabolic disruptions that are characteristic of obesity. However, the study results suggest that bromocriptine programming improved the redox status by protecting against nonalcoholic fatty liver disease, perhaps through SIRT1. The observations in this study further encourage additional studies to better understand the effects of bromocriptine during this critical life stage and its potential therapeutic use in preventing developmental diseases. Conflict of interest statement The authors have no financial and commercial conflicts of interest.
Acknowledgments All authors are grateful to Miss Monica Moura and Mr. Ulisses Risso Siqueira for technical assistance. This research was supported by the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico — CNPq), the Coordination for the Enhancement of Higher Education Personnel (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior —CAPES) and the State of Rio de Janeiro Carlos Chagas Filho Research Foundation (Fundacão Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro —FAPERJ).
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