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Swimming training prevents metabolic imprinting induced by hypernutrition during lactation
Clinical Nutrition ESPEN 10 (2015) e13ee20
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Clinical Nutrition ESPEN journal homepage: http://www.clinicalnutritionespen.com
Original article
Swimming training prevents metabolic imprinting induced by hypernutrition during lactation udia Regina Capriglioni Cancian a, Stefani Valeria Fischer a, *, Cla Elisangela Gueiber Montes a, Nayara de Carvalho Leite b, Sabrina Grassiolli a a b
Department of General Biology, State University of Ponta Grossa, Ponta Grossa, Parana, Brazil ~o Paulo, Brazil Department of Structural and Functional Biology, Institute of Biology, UNICAMP, Campinas, Sa
a r t i c l e i n f o
s u m m a r y
Article history: Received 6 January 2014 Accepted 29 October 2014
Background & aims: Reduction in litter size during lactation induces hypernutrition of the offspring culminating with altered metabolic programming during adult life. Overnourished rats present alterations in the endocrine pancreas and major predisposition to the development of type 2 diabetes. Our study evaluated the impact of swimming training on insulin secretion control in overnourished rats. Methods: At postnatal day 3 male rat pup litters were redistributed randomly into Small Litters (SL, 3 pups) or Normal Litters (NL, 9 pups) to induce early overfeeding during lactation. Both groups were subjected to swimming training (3 times/week/30 min) post-weaning (21 days) for 72 days. At 92 days of life pancreatic islets were isolated using collagenase technique and incubated with glucose in the presence or absence of acetylcholine (Ach, 0.1e1000 mM) or glucagon-like peptide 1 (GLP1, 10 nM). Adipose tissue depots (white and brown) and endocrine pancreas samples were examined by histological analysis. Food intake and body weight were measured. Blood biochemical parameters were also evaluated. Results: Swimming training prevented metabolic program alteration by hypernutrition during lactation. Exercise reduced obesity and hyperglycemia in overnourished rats. Pancreatic islets isolated from overnourished rats showed a reduction in glucose-induced insulin secretion and cholinergic responses while the insulinotropic action of GLP1 was increased. Physical training effectively restored glucoseinduced insulin secretion and GLP1-stimulated action in pancreatic islets from overnourished rats. However, swimming training did not correct the weak cholinergic response in pancreatic islets isolated from overnourished rats. Conclusions: Swimming training avoids obesity development, corrects glucose-induced insulin secretion, as well as, GLP1 insulinotropic response in overnourished rats. © 2014 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
Keywords: Metabolic program Islets Exercise Adipose tissue
1. Introduction Changes in the nutritional environment such as high lipid and carbohydrate consumption associated with a sedentary modern lifestyle are central elements of the obesity epidemic particularly during critical periods of development including the prenatal and neonatal periods. These phases are considered windows into early
* Corresponding author. Comandante Paulo Pinheiro Schimidht Street, 366, , Brazil. Tel.: þ55 42 99725626. Uvaranas, Ponta Grossa, Parana E-mail address: [email protected] (S.V. Fischer).
development processes during which alterations in the nutritional state could have lifelong consequences for the development of major diseases during adult life [1,2]. There are numerous studies showing that under- or overnutrition during gestation and/or lactation in rats affects the metabolic programming of the energetic metabolism and modulates insulin action and secretion from pancreatic islets in the adult life. These precocious dietary interventions are accompanied by adult-onset diseases in the offspring particularly obesity and type 2 diabetes (T2D) [3]. The definitive diagnosis of T2D is directly dependent upon the association of two conditions: insulin resistance and beta cell dysfunction. Beta cell failure in T2D involves
http://dx.doi.org/10.1016/j.clnme.2014.10.003 2212-8263/© 2014 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
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reduction of overall beta cell mass and insulin secretory function [4]. Several studies have shown that nutritional intervention during critical developmental periods of fetal and early post-natal life induce adverse effects on beta-cell development and glucoseinduced insulin secretion [1,5]. Experimental studies in rats have demonstrated that modifications in the litter size during lactation can induce early postnatal overnutrition. Animals reared in small litters (SL) are considered ‘overnourished’ and are more susceptible to obesity and metabolic disorders during adult life. These effects are attributed to large milk volume and greater energy intake during lactation. Rats from SL exhibit hyperphagia, obesity, elevated triacylglycerols, hyperleptinemia, hyperinsulinemia and impaired glucose tolerance during adulthood [2]. It has been shown that regular physical training reduces adipose tissue content, increases insulin sensitivity, and improves glucose homeostasis. However, the effects of physical training on the endocrine pancreas are contradictory [6,7]. In islets isolated from lean-exercised rats a reduction in glucose-induced insulin secretion was frequently observed. In contrast, islets isolated from obese and/ or diabetic exercised models exhibited variable effects on insulin secretion control [8,9]. In this study we investigated the impact of swimming training on obesity induced by overnutrition during lactation.
stress. According study of the Gobatto et al. the intensity of the swimming training can be determined by of the amount of load weight attached to the tail [10]. Using Maximal Lactate Steady State (MLSS) these authors have demonstrated that the use of loads higher than 6% of the body weight, in long lasting exercise sessions, at the beginning of a training period, could lead to undesirable effects of intense physical exercise. In this sense, Rocha et al., 2012 recently have demonstrated that higher intensity of swimming training could promotes cardiac lesions. Thus, load weight equivalent of 5% of the body weight was attached to the base of the tail to avoid acclimation and to guarantee a low at moderate intensity of exercise. In addition, Shima et al., 1993 showed that exercise training once every 2 and 3 days prevented the development of NIDDM in OLETF rats (a model of spontaneous non-insulin dependent diabetes mellitus) [11]. Contrary at other studies our swimming program was started precociously (21 days) and maintained until adult life (92 days). Scomparin et al., 2006 using similar swimming training program have demonstrated that fat reduction and metabolic adjusts are more accented when swimming training started after weaning [12]. Thus, 4 experimental groups were studied: NL-SED, NL-EXE, SL-SED and SL-EXE. A total of 15-20 rats were exercised according to their group assignment and were subjected to experimental procedures described in the next sections. All experimental protocols were performed 48 h after the last swimming session.
2. Material and methods 2.3. Islet isolation and incubation 2.1. Animals All animal protocols were approved by the Ethics Committee for Experimental Animals at the State University of Ponta Grossa (CEUA number 03482/2012) which based their analysis on the principles adopted and promulgated by Brazilian Law. Thirty virgin Wistar rats (age 70 ± 10 days) were mated with male rats using the harem system at a proportion of 3 females to 1 male. After mating, each female was placed in an individual cage with unlimited access to water and food. The day after delivery, excess pups in each litter were removed to retain 10 pups per dam. At postnatal day 3, male rat pup litters were redistributed randomly into Small Litters (SL, 3 pups) or Normal Litters (NL, 9 pups) to induce early overfeeding during lactation. According described by several authors (Babicky et al., 1973; Fiorotto et al., 1991; Plagemann et al., 1992), neonatal over-nutrition can be easily induced by a reduction of the number of pups of the litter during lactation. Thus, SL rat pups experience quantitative as well as qualitative over-nutrition during neonatal life once that, this manipulation results in a surplus of milk for each offspring accompanied by changes in the composition of the milk with increased caloric and fat content. A total of 15 litters were evaluated for each experimental group. Offspring were weaned at 21 days of age and were allowed free access to stock diet and water thereafter. After weaning, 6 rats per cage were maintained. To eliminate potential sex-related outcome variance, only male offspring were studied. All groups were maintained at a controlled temperature (21 ± 3 C) with a 12e12 h light-dark cycle, and food and water were provided ad libitum.
At 93 days old, 4e6 rats from each group were sacrificed and pancreatic islets were isolated with collagenase as previously described by Lacy and Kostianovsky [13], with a few adaptations. After anesthesia (xylazine and ketamine; 0.6 mg and 3 mg/100 g body weight, respectively), the abdominal wall was opened and 10 mL of Hank's buffered saline solution (HBSS) containing collagenase type V (1.0 mg/mL) was injected into the rat's common bile duct. The pancreas was quickly excised and incubated for 15 min at 37 C, and the suspension was subsequently filtered and washed with HBSS [0.1% bovine serum albumin fraction V (BSA)]. Islets were collected with the aid of a microscope. Batches of 4 islets were pre-incubated for 60 min in 1 mL of normal KrebseRinger solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 24 mM NaHCO3 and 5.6 mM glucose. This solution was gassed with O2/CO2 (95/5%) to maintain a pH of 7.4 and supplemented with BSA (0.1%). After adaptation to a low glucose concentration (5.6 mM) solution, islets were incubated for 60 additional min in the presence of stimulatory glucose concentrations of 8.3 mM, 16.7 mM, 20.0 mM and 28 mM. After pre-incubation, other islet batches were incubated for 60 min in Krebs solution containing glucose (8.3 mM) in the presence of acetylcholine at different concentrations (0.1, 1, 10, 100 and 1000 mM). Neostigmine (10 mM), an inhibitor of acetylcholinesterase activity, was used to avoid acetylcholine degradation. The effects of glucagon-like 1 peptide (GLP1) on glucose-induced insulin secretion were also investigated. In this experiment, islets were incubated with 1 mL of Krebs solution containing 5.6 mM or 16.7 mM glucose in the presence or absence of GLP1 (10 nM). Samples of incubation media were obtained and frozen until measurements for secreted insulin could be performed by radioimmunoassay (RIA).
2.2. Swimming training 2.4. Blood biochemical analyses After 21 days, SL and NL groups were subdivided into exercised (EXE) or sedentary (SED) animals. Swimming training was performed according to the protocol previously described by Gobatto et al [10]. and modified by Leite et al [8]. Briefly, exercised groups swam 3 times/week for 30 minutes for a period of 72 days. Water temperature was maintained at 32 ± 2 C to eliminate cold-induced
After 12 h of fasting other groups of rats (n ¼ 10/group) were killed by decapitation and total blood collected in heparinized tubes. Plasma glucose, cholesterol and triglyceride levels were measured by enzymatic procedures using commercial kits (Gold Analisa®, Belo Horizonte e MG, Brazil) and read by means of an
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automatic analyzer (Selectra II da Bayer). Insulin was measured via RIA. 2.5. Histology Part of the rats (n ¼ 3e4/group) used by biochemical parameters were also used by histological studies. The abdominal cavity of the rats was opened and several tissues were collected for histological analysis according to the procedures described hereafter. The splenic portion (tail) of the whole pancreas was rapidly removed and cleared of fat. The subcutaneous inguinal deposit and the visceral mesenteric and epididymal depots were collected from white adipose tissue. In addition, interscapular brown adipose tissue was also removed. All tissues were fixed in ALFAC solution (85 mL 80% alcohol, 10 mL 40% formaldehyde, and 5 mL glacial acetic acid) for 20e24 h at room temperature. After fixation, tissues were dehydrated in graded ethanol, cleared in xylol, and embedded in paraffin at 60 C. 5 mm sections were cut on the microtome (Leica RM 2025, Germany), stained with hematoxylin and eosin (H&E) and then analyzed with image analysis software. At least 3 slices per adipose tissue sample and 9 fields of view were examined for each slice to determine the adipocyte cross-sectional areas. The cell parameters were measured using an image analysis system (Image J 1.39f, NIH e Bethesda, MD, USA). The sectional areas of the adipocytes and islets were determined from digital images acquired at random (Olympus BX51 microscope, LC Evolution camera, TIFF format, 36-bit color, 1280 1024 pixels). A total of 50 adipocytes were measured per section and all islets were measured in each section. The number of adipocytes and islets was also evaluated. The number of nuclei was evaluated in the brown adipose tissue. Three representative areas were scored in each section, and the average values were used. 2.6. Food and water intake After weaning (21 days), rats from all groups were weighed, and their food and water intake was determined 3 times per week until adult life (93 days). Food and water intake were calculated as the amount consumed divided by the body weight of each animal and are expressed in g/100 g of body weight (bw). In this way, the total area under the curve (AUC) of food and water consumption versus time was calculated. All rats in the cage were of the same group and each rats was individually indentified by tattoo in the tail. 2.7. Obesity Adipose tissue accumulation was evaluated in all experimental groups to confirm obesity in adult life (93 days). Thus, same rats used in protocols described above were studied (n ¼ 10e14/group). The mesenteric and periepididymal deposits were weighed and designated as visceral adipose tissue, while inguinal adipose tissue was weighed and designated as subcutaneous deposits. In addition, interscapular brown adipose tissue was also measured. The weights of adipose tissues and organs were expressed as g/100 g bw. The Lee index [bw (g)1/3/nasal-anal length (cm)] was calculated as a predictor of obesity. During lactation we also evaluated daily the evolution of the body weight of the offspring and at weaning (21 days) the Lee Index of the offspring was also calculated. 2.8. Western blotting assay Isolated islets were pelleted and resuspended in buffer containing protease inhibitors as previously described by Amaral et al. [14]. Total protein content was determined by the Bradford (1976) method [15]. Fifteen (15) mg of total protein extract from pancreatic
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islets were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then incubated with Phospholipase C b (PLC b) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or Muscarinic Receptor 3 (M3) (Sigma Aldrich, St. Louis, Mo., USA) antibodies. Antibody dilutions were made according to the manufacturer's instructions in TRIS-Tween buffer containing 30 g/L of dried skim milk. Band detection was performed by chemiluminescence (Pierce Biotechnology, Rockford, IL) after incubation with a horseradish peroxidase-conjugated secondary antibody. Band intensities were quantified by optical densitometry (Image J, National Institutes of Health, USA). 2.9. Chemical and reagents Analytical grade reagents and deionized water were used. Collagenase type V, BSA (fraction V), acetylcholine and the inhibitor neostigmine were obtained from Sigma (St. Louis, Mo., USA). 2.10. Statistical analysis The results are shown as the mean ± the standard error of the mean (SEM). The data were subjected to Student's t -test or a one way analysis of variance (ANOVA) followed by a Bonferroni posttest analysis with selected pairs grouped when appropriate. P values less than 0.05 (p < 0.05) were considered statistically significant. The statistical tests were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). 3. Results Hypernutrition during lactation induced obesity and hyperglycemia during adulthood in rats according to the results in Table 1. SL-SED rats presented with an increase of 8.7% and 3% in body weight and LI, respectively, when compared to NL-SED animals. In addition, basal glycemia was 16.2% higher in SL-SED rats than in NLSED animals (p < 0.05). The insulin levels, total cholesterol, triglyceride levels and nasal-anal lengths were not significantly different in SL-SED rats. The effects of swimming training in both experimental groups are also represented in Table 1. Both of the exercised groups (NL-EXE and SL-EXE) presented with a reduction of 8.8% and 7.2% in body weight, respectively, when compared to the sedentary groups. Similarly, the LI was decreased by 2.8% and 3.7% in NL-EXE and SL-EXE rats, respectively, when compared to the sedentary groups. In addition, NL-EXE and SL-EXE rats exhibited a reduction of approximately 16% and 20%, respectively, in glycemia, total cholesterol and triglycerides when compared to the NL-SED and SL-SED groups. Fig. 1(A-E) shows body weight, LI, food intake and hydric intake evolution. During lactation (0e21 days), SL rats showed an increase in body weight evolution when compared to NL rats (Fig. 1A). This difference was first observed at postnatal day 12 and persisted for 21 days. Thus, during the lactation phase the AUC of the body weight was increased by 18.5% in SL rats when compared to the NL group (p < 0.05). The LI was also approximately 9.1% higher in SL rats than in NL rats at 21 days (Fig. 1B). Body weight evolution postweaning (22e90 days) is shown in Fig. 1C. SL-SED rats exhibited a slight increase in body weight evolution when compared to NL-SED rats. The AUC of body weight measurements post-weaning was 9.5% higher in SL-SED rats than in NL-SED animals. However, during this period body weight gain was not associated with changes in food or water intake (Fig. 1D and E). In the NL-EXE and SL-EXE groups, swimming training reduced the body weight AUC by 6.7% and 8%, respectively, when compared to the NL-SED and SL-SED rats. In both experimental groups, swimming training did not alter food or water intake.
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Table 1 Effects of swimming training in the biometric and biochemistry parameters in overnourished rats. NL-SED Body weight (g) Lee index Naso-anal length (cm) Basal glucose (mg/dL) Basal insulin (ng/mL) Total cholesterol (mg/dL) Triglycerides (mg/dL)
308.00 0.309 21.77 98.57 0.22 128.40 63.69
± ± ± ± ± ± ±
NL-EXE 3.87b,c 0.002b,c 0.14 4.22b,c 0.03 3.09b 3.90b
282.40 0.300 21.86 83.25 0.18 110.00 51.10
± ± ± ± ± ± ±
SL-SED 3.90a 0.001a 0.14 3.43a 0.03 2.89a 2.68a
335.00 0.318 22.05 114.5 0.29 136.50 64.84
SL-EXE ± ± ± ± ± ± ±
4.41a,d 0.002a,d 0.18 4.27a,d 0.04 8.30d 4.52d
310.70 0.306 22.10 96.35 0.27 100.40 52.45
± ± ± ± ± ± ±
2.90c 0.002c 0.13 5.21c 0.03 2.68c 2.60c
The data are expressed as the mean ± SEM obtained from 15e20 rats by group. Letters above the numbers represent a significant difference of p < 0.05 obtained by analyses of variance ANOVA followed by Bonferroni's posttest (selected par groups). a NL-SED. b NL-EXE. c SL-SED. d SL-EXE.
The impact of hypernutrition and swimming training on adipose tissue content and adipocyte hypertrophy is shown in Fig. 2. The SLSED groups exhibited increases in white visceral adipose tissue when compared to NL-SED rats. The mesenteric (Fig. 1-L) and retroperitoneal (Figure E-H) content were approximately 25%
higher in SL-SED rats than in NL-SED groups. However, subcutaneous inguinal fat and brown adipose tissue content were similar between SL-SED and NL-SED rats. The visceral white adipose tissue content was reduced by approximately 20% in NL-EXE rats when compared to the NL-SED group. Similarly, swimming training
Fig. 1. Effects of swimming training on body weight and food and water intake of overnourished rats. Values are expressed as the mean ± SEM of 15e20 rats for all groups. Body weight and ìndice Lee was evaluated during lactation (1 at 21 days, Fig. 1A and B respectively) and after weaning (22 at 90 days, Fig. 1C). The food and water intake are represented in the lower panels (Fig. 1D and E). The respective areas under the curve (AUC) are shown in the insets. * represents a significant difference of p < 0.05 between the NL and SL groups obtained by Student's t test. Letters above the bars show significant differences of p < 0.05 obtained by an ANOVA followed by Bonferroni's posttest (selected par groups). *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE.
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Fig. 2. White and Brown adipose tissue content and morphological aspects of SL-exercised rats. Representative photomicrographs of white visceral adipocytes (Fig. 2A-L), subcutaneous adipocytes (Fig. 2M-P) and brown adipocytes (Fig. 2Q-T). The photomicrographs were assessed by H&E staining (200 and 400, white and brown adipocytes, respectively) with scale bars indicating 50 mm and 20 mm. The diameter and number of white adipocytes were evaluated. In brown adipocytes, proliferation was evaluated by the number of nuclei. Respective differences between groups are described in the results section. The lower figures (2U-2Y) represent white and brown adipose tissue weights. Values are expressed as the mean ± SEM of 15e20 rats for all groups. Letters above numbers show significant differences of p < 0.05 obtained by an ANOVA followed by Bonferroni's posttest (selected par groups). *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE.
reduced white visceral adipose tissue content by approximately 19% in SL-EXE animals when compared to SL-SED rats (p < 0.05). In both groups, subcutaneous inguinal fat (Figure M-P) content was not affected by swimming training. In contrast, brown adipose tissue content increased 106.8% and 63.9% for the NL-EXE and SLEXE groups, respectively, when compared to their sedentary counterparts (p < 0.05). Hypernutrition during lactation induces hypertrophy in white adipocyte tissue according to the morphological data presented in Fig. 2 and Table 3. Adipocytes isolated from epididymal, retroperitoneal and inguinal fat contained approximately 20% more adipocytes in SL-SED rats when compared to NL-SED animals (p < 0.05). Adipocyte areas from mesenteric fat were similar between SL-SED and NL-SED rats. Only epididymal fat content exhibited a reduction of 15% in adipocyte number in SL-SED rats when compared to NL-SED animals. Similarly, the number of adipocytes in brown adipose tissue exhibited a reduction of 25.4% in the SL-SED group when compared to NL-SED rats (p < 0.05). Swimming training reduced the area of the adipocytes by approximately 16% in epididymal and retroperitoneal fat, 23.3% in mesenteric fat and 8.6% in inguinal fat in NL-EXE groups when compared to NL-SED animals. Similarly, in SL-EXE rats the area of the adipocytes was reduced by approximately 18.5% in epididymal
deposits, 32.4% in retroperitoneal deposits and 13.6% in inguinal fat deposits when compared to adipocytes isolated from SL-SED rats (p < 0.05). Glucose-induced insulin secretion and pancreatic islet morphology in SL rats subjected to swimming training are shown in Fig. 3. Pancreatic islets from SL-SED rats presented with a reduction in glucose-induced insulin secretion. In the presence of high glucose concentrations, pancreatic islets isolated from SL-SED rats exhibited a reduction in glucose-induced insulin secretion of approximately 33% when compared to islets obtained from the NLSED groups. Swimming training caused a reduction in glucoseinduced insulin secretion in pancreatic islets from NL-EXE rats when compared to islets isolated from the NL-SED group. In contrast, pancreatic islets from SL-EXE rats exhibited an increase in glucose-induced insulin secretion of approximately 95% when compared to islets isolated from SL-SED animals (p < 0.05). Pancreatic islets isolated from SL-SED animals exhibited reductions in area and number of 34.2% and 30%, respectively, when compared to islets isolated from NL-SED rats (p < 0.05). In both groups, swimming training altered the amount of proliferation and the size of the pancreatic islets. The NL-EXE and SL-EXE groups exhibited an increase in pancreatic islet number of 31.7% and 39.6%, respectively, when compared to their sedentary counterparts. In addition, islet
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Fig. 3. Glucose-induced insulin secretion and histological analysis of pancreatic islets isolated from SL-exercised rats. Pancreatic islets were isolated by the collagenase technique as described in the methods section and incubated in the presence of different glucose concentrations (Fig. 3A). Values are expressed as the mean ± SEM of 20e24 observations for each glucose condition. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SLEXE. Fig. 3B shows representative photomicrographs of pancreatic islets assessed by H&E staining (400) with scale bars indicating 200 mm. The diameter and number of pancreatic islets were evaluated. Individual islet profiles are shown in the insets. Respective data are described in the results section.
area was reduced by 41.1% and 39.3% in the NL-EXE and SL-EXE groups, respectively, when compared to islets isolated from the sedentary groups (p < 0.05). The effects of hypernutrition and swimming training on cholinergic-induced potentiation and islet protein expression were also evaluated (Fig. 4A-C). The addition of Ach resulted in a dosedependent increase in insulin secretion from islets isolated from
Fig. 4. Cholinergic insulinotropic response of islets isolated from overnourished rats subjected to swimming training (A) and expression of M3 (B) and PLC b (C). Groups of 4 islets were incubated in 1 mL of Krebs solution with glucose (8.3 mM) in the presence of different concentrations of acetylcholine. The data are shown relative to 100% (only glucose concentration is 8.3 mM). Four or six rats were sacrificed from each group to form a pool of islets. Values are expressed as the mean ± SEM of 20e24 observations for each acetylcholine condition. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE. Representative western blotting images are shown and densitometry quantification is indicated by vertical bars for M3 and PLC b for all groups. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. DNL-SED versus NL-EXE.
the NL-SED group. In these islets, high Ach concentrations (such as 100 and 1000 mM) increased insulin secretion by approximately 6 fold (p < 0.05). Pancreatic islets isolated from the NL-EXE group exhibited an attenuated Ach insulinotropic effect, which was approximately 50% less than that observed in islets isolated from NL-SED rats. Hypernutrition during lactation altered the cholinergic-induced potentiation of pancreatic islets. In the presence of low or moderate Ach concentrations (0.1e10 mM) the cholinergic potentiation effect was inhibited (by approximately 104%) in islets isolated from the SL-SED group, whereas cholinergic-induced effects were observed only in the presence of the highest doses of Ach (100e1000 mM). Swimming training accentuated the inhibitory response of Ach in islets isolated from the SL-EXE group (p < 0.05). Muscarinic receptor (M3) and PLCb expression of the isolated pancreatic islets are represented in Fig. 4B and C, respectively. There was a reduction in M3 expression in the NL-EXE group (of 44.8%) when compared to NL-SED rats (p < 0.05). The expression of PLCb was not affected by hypernutrition or exercise training. The insulinotropic effects of GLP1 on glucose-induced insulin secretion are shown in Fig. 5(A and B). In the presence of low
Fig. 5. GLP-1 stimulated action of glucose-induced insulin secretion in islets isolated from overnourished rats subjected to swimming training. Groups of 4 islets were incubated in 1 mL of Krebs solution with 5.6 mM glucose (Fig. 5A) or 16.7 mM glucose (Fig. 5B) in the presence of GLP-1 (10 nM). The data are shown relative to 100% (5.6 or 16.7 mM glucose only controls). Four or six rats were sacrificed from each group to form a pool of islets. Values are expressed as the mean ± SEM of 20e24 observations for each glucose condition. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE.
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glucose (5.6 mM), insulin secretion increased by 73% and 158% in islets isolated from NL-SED and SL-SED rats, respectively. Under these glucose conditions, both exercised groups exhibited a reduction in the GLP1 insulinotropic effect (Fig. 5A, p < 0.05). However, in the presence of a high glucose concentration (16.7 mM), GLP1-stimulated action was observed only in islets isolated from SL rats. The insulinotropic effect of GLP1 was 260% and 115% in islets isolated from SL-SED and SL-EXE rats, respectively (Fig. 5B, p < 0.05). 4. Discussion Reduction in litter size during the suckling period can induce nutritional programming that alters the metabolism of offspring during adult life [2]. Postnatal suckling period is a vulnerable period from offspring because in this phase many organs, as well as, important energetic hypothalamic pathways are established [16]. Consistent with these reports, in our study pups assigned to small litter showed an increase in body weight gain and LI during suckling period remaining overweight at 93-old-days. Similarly in humans, several observations have shown that rapid weight gain in the early stages predispose at overweight as well as the future development of obesity in adulthood [17]. Our study also demonstrated that in SL-adult rats the increase in body weight gain was accomplished of rises in fat body content and fasting hyperglycemia. It has been demonstrated that reduction in the litter size during lactation promotes overweight, glucose tolerance and hyperphagia in adulthood [2,18]. However, in our study was not observed hyperphagia in SL-adult rats. The reason behind these inconsistent results could involve differences in food intake expression according previously established by Xiao et al [19]. In our study we also did not observe dyslipidemia or hyperinsulinemia in SL-adult rats, corroborating with results obtained by Rodrigues et al. with SL-rats of the same age [18]. Independently these divergent data, in our study the obesity in SL-adult rats can be confirmed by high white adipose tissue content accompanied by adipocyte hypertrophy. In addition, we also demonstrated that hypernutrition during lactation increase the size and number of lipid droplets in brown adipose tissue from SL-adult rats, suggesting reduction in brown adipose tissue thermogenesis. This confirm results obtained by Xiao et al. in SL-rats [19]. Our swimming training was started at 21 days, in this age SLrats already presented heavier body weight and increased LI. Chronic swimming training effectively avoided obesity, hiperglycaemia, reduced lipid levels and adipose tissue content in SLtrained rats. Thus, our study demonstrated by the first time that SL-rats are responsive at benefits of the aerobic exercise program started immediately after weaning and maintained until adult life. These data also corroborated with study realized by Scomparin et al. showing that precocity of the exercise training is important to avoid obesity abnormalities [12]. The development of the endocrine pancreas is vulnerable to nutritional insults during the immediate post-natal phase [1]. Pancreatic endocrine dysfunction is associated with a reduction in beta cell mass and impaired glucose-stimulated insulin secretion [4]. In our study, glucose-induced insulin secretion and islets number were reduced in endocrine pancreas from SL-adult rats. Similar results were obtained by Waterland and Garza. [1]. According with these authors the endocrine pancreas is the primary tissue affected by metabolic imprinting. The central aspect of our study was to evaluate the influence of swimming training on the endocrine pancreas of SL-adult rats. Interestingly, we observed that swimming training was able to restore glucose responsiveness in islets isolated from SL-adult rats. A study performed by Dela et al. showed that physical training
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increased glucose-induced insulin secretion in pancreatic islets isolated from diabetic patients [6]. According to the authors, this pancreatic response to exercise is directly dependent upon beta cell secretory capacity and diverges in health and diabetic conditions. In addition, we also demonstrated that swimming training promoted increases in islet numbers and a simultaneous reduction of islet size in pancreas samples isolated from SL-adult rats. Shima et al. showed that physical training improves islet morphological alterations observed in Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats with T2D [11]. Similarly, Park et al. demonstrated that exercise can stimulate pathways of proliferation in islets of diabetic rats [20]. Taken together, these results indicate that swimming training started at the post-weaning stage and maintained during development can avoid abnormalities in glucose responsiveness induced by hypernutrition during suckling. Disruption in cholinergic insulinotropic action is directly involved in pancreatic islet dysfunction [21,22]. Cholinergic effects on pancreatic beta cells are exerted primarily through muscarinic receptors (MRs). The presence of multiple MRs (1e4) has been demonstrated in pancreatic islets or beta cells [23], however MR subtype 3 (MR3) appears to be the predominant subtype expressed by pancreatic beta cells [22]. For the first time, our study evaluated the Ach response of pancreatic islets isolated from overnourished rats. A cholinergic inhibitory response was observed in islets isolated from SL-adult rats at low concentrations of Ach, whereas a weak insulinotropic effect was observed at high concentrations of Ach. Similarly, pancreatic islets isolated from undernourished rats (a model of metabolic programming) exhibited a reduction in MR3 activation [24]. Moreover, studies have also demonstrated that Ach can inhibit glucose-induced insulin secretion via MR subtype 2 or 4 (MR2 or 4) [23,25]. Thus, we suggest that altered composition of MR may be involved in these abnormal cholinergic responses in pancreatic islets isolated from SL-rats because MR3 or PLCb expression did not change in this group of islets. This possibility was recently confirmed by de Oliveira et al. in islets isolated from undernourished rats [26]. Surprisingly, our results also demonstrate that swimming training accentuated the inhibitory action of Ach on islets obtained from both groups. In islets obtained from lean rats, these responses seem to involve a reduction in MR3 expression. Contrary data were obtained by Almeida et al. In this study, lean trained animals submitted to an acute exercise regimen increased their insulin secretion stimulated by the cholinergic agent carbachol [7]. Divergent achieves could be attributed at acute or chronic effect of the exercise training. Thus, we suggested that weak cholinergic insulinotropic effect in islets from exercised rats could be the result of an adaptation to avoid hypoglycemia once that physical training also improves insulin sensitivity. Finally, we also observed that islets from SL-adult rats exhibit increased GLP1 insulinotropic action. GLP-1 can promote glucosestimulated insulin secretion and stimulates pancreatic beta cell masses [27]. This incretin hormone is primarily produced by intestinal L cells; however, recent evidence indicates that GLP1 may also be produced in the islets [28]. There are no known studies that evaluate GLP1 action or production in SL-overnourished rats. However, high-carbohydrate (HC) milk formula offered during the suckling period can also induce hypernutrition [5]. Similar to our results, islets isolated from HC rats exhibit increased insulinotropic GLP1 responses. In addition, Miguel et al. have demonstrated the cooperative insulinotropic effects of Ach and GLP1 in a beta tumor cell line [29]. Thus, we suggest that the high GLP1 insulinotropic action observed in islets isolated from overnourished rats could be provoked by altered GLP1 expression in pancreatic islets and/or by the altered cholinergic effects demonstrated by our results. Our study also demonstrates that swimming training reduced GLP1 insulinotropic action at low glucose concentrations. Similar results
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were obtained by Svidnicki et al. in pancreatic islets isolated from hypothalamic obese rats subjected to swimming training [30]. These results seem to indicate that physical training can correct GLP1 insulinotropic action in pancreatic beta cells isolated from overnourished rats. This study demonstrates that early swimming training can prevent obesity installation, correct glucose-induced insulin secretion, as well as, GLP1 insulinotropic response in pancreatic islets isolated from overnourished rats without altering the abnormal cholinergic response. Strengths and Limitations: Our research shows clearly that the exercise introduced early during development is capable of avoids the deleterious effects induced by metabolic program. This aspect is of the particular interest to blockage obesity progression among children, once that, research into the 'developmental origins of health and disease' (DOHaD) has now firmly established that preand perinatal developmental perturbations can predispose to obesity in adult life. However, our study also presents limitations. The effect of metabolic program is sex-dependent, our study evaluated only male rats, thus we cannot affirm that similar effects would be observed in female. In addition, more adequate technique is necessary to evaluate exercise program (time, intensity and frequency) before of extrapolates to early childhood development. Statement of authorship Sabrina Grassiolli and Stefani Valeria Fisher designed the udia Regina Capriglioni Cancian, research. Stefani Valeria Fisher, Cla and Elisangela Gueiber Monte, conducted the research and analyzed the data. Nayara de Carvalho Leite performed the molecular techniques. Stefani Valeria Fisher and Sabrina Grassiolli wrote the paper. Stefani Valeria Fisher assumes primary responsible for this manuscript's final content. All authors read and approved the final manuscript. Conflict of interest statement and funding sources None of the authors declare a conflict of interest. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. We are grateful to Coor~o de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) denaça for the scholarship offered to Post-Graduate Student Stefani Valeria Fisher. Acknowledgments The authors would like to thank Professor Paulo Cesar de Freitas Mathias and Julio Cesar de Oliveira from Universidade Estadual de (UEM) e Parana e Brasil for assistance with the radioimMaringa ~es Carneiro munoassay technique and Professor Everardo Magalha from Universidade Estadual de Campinas e Campinas e Brasil for molecular technique support. References [1] Waterland RA, Garza C. Early postnatal nutrition determines adult pancreatic glucose-responsive insulin secretion and islet gene expression in rats. J Nutr 2002;132(3):357e64. €tz F, Rohde W, Do €rner G. Obesity and enhanced [2] Plagemann A, Heidrich I, Go diabetes and cardiovascular risk in adult rats due to early postnatal overfeeding. Experimental and Clinical Endocrinology & Diabetes 1992;99(03):154e8. [3] Cottrell EC, Ozanne SE. Developmental programming of energy balance and the metabolic syndrome. Proc Nutr Soc 2007;66(2):198e206. [4] Weir GC, Bonner-Weir S. Five stages of evolving beta-cell dysfunction during progression to diabetes. Diabetes 2004;53(Suppl. 3):S16e21. [5] Srinivasan M, Aalinkeel R, Song F, Patel MS. Programming of islet functions in the progeny of hyperinsulinemic/obese rats. Diabetes 2003;52(4):984e90.
[6] Dela F, Michael E, Mikines KJ, Galbo H. Physical training may enhance b-cell function in type 2 diabetes. Am J Physiol Endocrinol Metab 2004;287(5): E1024e31. [7] Almeida FN, Proença AR, Chimin P, Marçal AC, Bessa-Lima F, Carvalho CR. Physical exercise and pancreatic islets: acute and chronic actions on insulin secretion. Islets 2012;4(4):296e301. [8] Leite NC, Ferreira TR, Rickli S, Borck PC, De Freitas Mathias PC, De Oliveira Emilio HR, et al. Glycolytic and mitochondrial metabolism in pancreatic islets from MSG-treated obese rats subjected to swimming training. Cell Physiol Biochem 2013;31(2e3):242e56. cary S, Peyot M-L, Latour MG, Lamontagne J, [9] Delghingaro-Augusto V, De Paradis-Isler N, et al. Voluntary running exercise prevents b-cell failure in susceptible islets of the Zucker diabetic fatty rat. Am J Physiol Endocrinol Metab 2012;302(2):E254e64. [10] Gobatto CA, Mello MaR De, Sibuya CY, De Azevedo JRM, Dos Santos LA, Kokubun E. Maximal lactate steady state in rats submitted to swimming exercise. Comp Biochem Physiol A Mol Integr Physiol 2001;130(1):21e7. [11] Shima K, Shi K, Sano T, Iwami T, Mizuno A, Noma Y. Is exercise training effective in preventing diabetes mellitus in the Otsuka-Long-EvansTokushima fatty rat, a model of spontaneous non-insulin-dependent diabetes mellitus? Metabolism 1993;42(8):971e7. [12] Scomparin D, Grassiolli S, Marçal A, Gravena C, Andreazzi A, Mathias P. Swim training applied at early age is critical to adrenal medulla catecholamine content and to attenuate monosodium L-glutamate-obesity onset in mice. Life Sci 2006;79(22):2151e6. [13] Lacy PE, Kostianovsky M. Method for the isolation of intact islets of langerhans from the rat pancreas. Diabetes 1967;16(1):35e9. [14] Amaral M, Ueno M, Carvalheira J, Carneiro E, Velloso L, Saad M, et al. Prolactinsignal transduction in neonatal rat pancreatic islets and interaction with the insulin-signaling pathway. Horm Metab Res 2003;35(05):282e9. [15] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72(1):248e54. [16] Habbout A, Li N, Rochette L, Vergely C. Postnatal overfeeding in rodents by litter size reduction induces major short-and long-term pathophysiological consequences. J Nutr 2013;143(5):553e62. [17] Stettler N, Stallings VA, Troxel AB, Zhao J, Schinnar R, Nelson SE, et al. Weight gain in the first week of life and overweight in adulthood a cohort study of European American subjects fed infant formula. Circulation 2005;111(15): 1897e903. Da Silva SV, Rodrigues DS, Nascimento AB, Barja[18] Rodrigues AL, De Souza EP, Fidalgo C, et al. Low expression of insulin signaling molecules impairs glucose uptake in adipocytes after early overnutrition. J Endocrinol 2007;195(3): 485e94. [19] Xiao XQ, Williams SM, Grayson BE, Glavas MM, Cowley MA, Smith MS, et al. Excess weight gain during the early postnatal period is associated with permanent reprogramming of brown adipose tissue adaptive thermogenesis. Endocrinology 2007;148(9):4150e9. [20] Park S, Hong SM, Lee JE, Sung SR. Exercise improves glucose homeostasis that has been impaired by a high-fat diet by potentiating pancreatic b-cell function and mass through IRS2 in diabetic rats. J Appl Physiol 2007;103(5): 1764e71. [21] Young J. Developmental origins of obesity: a sympathoadrenal perspective. Int J Obes 2006;30:S41e9. [22] Gautam D, Han SJ, Duttaroy A, Mears D, Hamdan F, Li J, et al. Role of the M3 muscarinic acetylcholine receptor in b-cell function and glucose homeostasis. Diabetes Obes Metab 2007;9(s2):158e69. [23] Miguel JC, Abdel-Wahab YH, Mathias PC, Flatt PR. Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulinsecreting b cell line. Biochim Biophys Acta (BBA) General Subjects 2002;1569(1):45e50. [24] Batista TM, Ribeiro RA, Amaral AG, De Oliveira CA, Boschero AC, Carneiro EM. Taurine supplementation restores glucose and carbachol-induced insulin secretion in islets from low-protein diet rats: involvement of Ach-M3R, Synt 1 and SNAP-25 proteins. J Nutr Biochem 2012;23(3):306e12. [25] Grassiolli S, Gravena C, De Freitas Mathias PC. Muscarinic M2 receptor is active on pancreatic islets from hypothalamic obese rat. Eur J Pharmacol 2007;556(1):223e8. [26] Oliveira J, Scomparin D, Andreazzi A, Branco R, Martins A, Gravena C, et al. Metabolic imprinting by maternal protein malnourishment impairs vagal activity in adult rats. J Neuroendocrinol 2011;23(2):148e57. [27] Drucker DJ. Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care 2003;26(10):2929e40. [28] Marchetti P, Lupi R, Bugliani M, Kirkpatrick C, Sebastiani G, Grieco F, et al. A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets. Diabetologia 2012;55(12):3262e72. [29] Miguel JC, Abdel-Wahab YH, Green BD, Mathias PC, Flatt PR. Cooperative enhancement of insulinotropic action of GLP-1 by acetylcholine uncovers paradoxical inhibitory effect of beta cell muscarinic receptor activation on adenylate cyclase activity. Biochem Pharmacol 2003;65(2):283e92. [30] Svidnicki PV, Leite NDC, Venturelli AC, Camargo RL, Vicari MR, Almeida MC, et al. Swim training restores glucagon-like peptide-1 insulinotropic action in pancreatic islets from monosodiumglutamate-obese rats. Acta Physiol 209(1) 2013:34e44.
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Original article
Swimming training prevents metabolic imprinting induced by hypernutrition during lactation udia Regina Capriglioni Cancian a, Stefani Valeria Fischer a, *, Cla Elisangela Gueiber Montes a, Nayara de Carvalho Leite b, Sabrina Grassiolli a a b
Department of General Biology, State University of Ponta Grossa, Ponta Grossa, Parana, Brazil ~o Paulo, Brazil Department of Structural and Functional Biology, Institute of Biology, UNICAMP, Campinas, Sa
a r t i c l e i n f o
s u m m a r y
Article history: Received 6 January 2014 Accepted 29 October 2014
Background & aims: Reduction in litter size during lactation induces hypernutrition of the offspring culminating with altered metabolic programming during adult life. Overnourished rats present alterations in the endocrine pancreas and major predisposition to the development of type 2 diabetes. Our study evaluated the impact of swimming training on insulin secretion control in overnourished rats. Methods: At postnatal day 3 male rat pup litters were redistributed randomly into Small Litters (SL, 3 pups) or Normal Litters (NL, 9 pups) to induce early overfeeding during lactation. Both groups were subjected to swimming training (3 times/week/30 min) post-weaning (21 days) for 72 days. At 92 days of life pancreatic islets were isolated using collagenase technique and incubated with glucose in the presence or absence of acetylcholine (Ach, 0.1e1000 mM) or glucagon-like peptide 1 (GLP1, 10 nM). Adipose tissue depots (white and brown) and endocrine pancreas samples were examined by histological analysis. Food intake and body weight were measured. Blood biochemical parameters were also evaluated. Results: Swimming training prevented metabolic program alteration by hypernutrition during lactation. Exercise reduced obesity and hyperglycemia in overnourished rats. Pancreatic islets isolated from overnourished rats showed a reduction in glucose-induced insulin secretion and cholinergic responses while the insulinotropic action of GLP1 was increased. Physical training effectively restored glucoseinduced insulin secretion and GLP1-stimulated action in pancreatic islets from overnourished rats. However, swimming training did not correct the weak cholinergic response in pancreatic islets isolated from overnourished rats. Conclusions: Swimming training avoids obesity development, corrects glucose-induced insulin secretion, as well as, GLP1 insulinotropic response in overnourished rats. © 2014 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
Keywords: Metabolic program Islets Exercise Adipose tissue
1. Introduction Changes in the nutritional environment such as high lipid and carbohydrate consumption associated with a sedentary modern lifestyle are central elements of the obesity epidemic particularly during critical periods of development including the prenatal and neonatal periods. These phases are considered windows into early
* Corresponding author. Comandante Paulo Pinheiro Schimidht Street, 366, , Brazil. Tel.: þ55 42 99725626. Uvaranas, Ponta Grossa, Parana E-mail address: [email protected] (S.V. Fischer).
development processes during which alterations in the nutritional state could have lifelong consequences for the development of major diseases during adult life [1,2]. There are numerous studies showing that under- or overnutrition during gestation and/or lactation in rats affects the metabolic programming of the energetic metabolism and modulates insulin action and secretion from pancreatic islets in the adult life. These precocious dietary interventions are accompanied by adult-onset diseases in the offspring particularly obesity and type 2 diabetes (T2D) [3]. The definitive diagnosis of T2D is directly dependent upon the association of two conditions: insulin resistance and beta cell dysfunction. Beta cell failure in T2D involves
http://dx.doi.org/10.1016/j.clnme.2014.10.003 2212-8263/© 2014 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
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reduction of overall beta cell mass and insulin secretory function [4]. Several studies have shown that nutritional intervention during critical developmental periods of fetal and early post-natal life induce adverse effects on beta-cell development and glucoseinduced insulin secretion [1,5]. Experimental studies in rats have demonstrated that modifications in the litter size during lactation can induce early postnatal overnutrition. Animals reared in small litters (SL) are considered ‘overnourished’ and are more susceptible to obesity and metabolic disorders during adult life. These effects are attributed to large milk volume and greater energy intake during lactation. Rats from SL exhibit hyperphagia, obesity, elevated triacylglycerols, hyperleptinemia, hyperinsulinemia and impaired glucose tolerance during adulthood [2]. It has been shown that regular physical training reduces adipose tissue content, increases insulin sensitivity, and improves glucose homeostasis. However, the effects of physical training on the endocrine pancreas are contradictory [6,7]. In islets isolated from lean-exercised rats a reduction in glucose-induced insulin secretion was frequently observed. In contrast, islets isolated from obese and/ or diabetic exercised models exhibited variable effects on insulin secretion control [8,9]. In this study we investigated the impact of swimming training on obesity induced by overnutrition during lactation.
stress. According study of the Gobatto et al. the intensity of the swimming training can be determined by of the amount of load weight attached to the tail [10]. Using Maximal Lactate Steady State (MLSS) these authors have demonstrated that the use of loads higher than 6% of the body weight, in long lasting exercise sessions, at the beginning of a training period, could lead to undesirable effects of intense physical exercise. In this sense, Rocha et al., 2012 recently have demonstrated that higher intensity of swimming training could promotes cardiac lesions. Thus, load weight equivalent of 5% of the body weight was attached to the base of the tail to avoid acclimation and to guarantee a low at moderate intensity of exercise. In addition, Shima et al., 1993 showed that exercise training once every 2 and 3 days prevented the development of NIDDM in OLETF rats (a model of spontaneous non-insulin dependent diabetes mellitus) [11]. Contrary at other studies our swimming program was started precociously (21 days) and maintained until adult life (92 days). Scomparin et al., 2006 using similar swimming training program have demonstrated that fat reduction and metabolic adjusts are more accented when swimming training started after weaning [12]. Thus, 4 experimental groups were studied: NL-SED, NL-EXE, SL-SED and SL-EXE. A total of 15-20 rats were exercised according to their group assignment and were subjected to experimental procedures described in the next sections. All experimental protocols were performed 48 h after the last swimming session.
2. Material and methods 2.3. Islet isolation and incubation 2.1. Animals All animal protocols were approved by the Ethics Committee for Experimental Animals at the State University of Ponta Grossa (CEUA number 03482/2012) which based their analysis on the principles adopted and promulgated by Brazilian Law. Thirty virgin Wistar rats (age 70 ± 10 days) were mated with male rats using the harem system at a proportion of 3 females to 1 male. After mating, each female was placed in an individual cage with unlimited access to water and food. The day after delivery, excess pups in each litter were removed to retain 10 pups per dam. At postnatal day 3, male rat pup litters were redistributed randomly into Small Litters (SL, 3 pups) or Normal Litters (NL, 9 pups) to induce early overfeeding during lactation. According described by several authors (Babicky et al., 1973; Fiorotto et al., 1991; Plagemann et al., 1992), neonatal over-nutrition can be easily induced by a reduction of the number of pups of the litter during lactation. Thus, SL rat pups experience quantitative as well as qualitative over-nutrition during neonatal life once that, this manipulation results in a surplus of milk for each offspring accompanied by changes in the composition of the milk with increased caloric and fat content. A total of 15 litters were evaluated for each experimental group. Offspring were weaned at 21 days of age and were allowed free access to stock diet and water thereafter. After weaning, 6 rats per cage were maintained. To eliminate potential sex-related outcome variance, only male offspring were studied. All groups were maintained at a controlled temperature (21 ± 3 C) with a 12e12 h light-dark cycle, and food and water were provided ad libitum.
At 93 days old, 4e6 rats from each group were sacrificed and pancreatic islets were isolated with collagenase as previously described by Lacy and Kostianovsky [13], with a few adaptations. After anesthesia (xylazine and ketamine; 0.6 mg and 3 mg/100 g body weight, respectively), the abdominal wall was opened and 10 mL of Hank's buffered saline solution (HBSS) containing collagenase type V (1.0 mg/mL) was injected into the rat's common bile duct. The pancreas was quickly excised and incubated for 15 min at 37 C, and the suspension was subsequently filtered and washed with HBSS [0.1% bovine serum albumin fraction V (BSA)]. Islets were collected with the aid of a microscope. Batches of 4 islets were pre-incubated for 60 min in 1 mL of normal KrebseRinger solution containing 120 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 24 mM NaHCO3 and 5.6 mM glucose. This solution was gassed with O2/CO2 (95/5%) to maintain a pH of 7.4 and supplemented with BSA (0.1%). After adaptation to a low glucose concentration (5.6 mM) solution, islets were incubated for 60 additional min in the presence of stimulatory glucose concentrations of 8.3 mM, 16.7 mM, 20.0 mM and 28 mM. After pre-incubation, other islet batches were incubated for 60 min in Krebs solution containing glucose (8.3 mM) in the presence of acetylcholine at different concentrations (0.1, 1, 10, 100 and 1000 mM). Neostigmine (10 mM), an inhibitor of acetylcholinesterase activity, was used to avoid acetylcholine degradation. The effects of glucagon-like 1 peptide (GLP1) on glucose-induced insulin secretion were also investigated. In this experiment, islets were incubated with 1 mL of Krebs solution containing 5.6 mM or 16.7 mM glucose in the presence or absence of GLP1 (10 nM). Samples of incubation media were obtained and frozen until measurements for secreted insulin could be performed by radioimmunoassay (RIA).
2.2. Swimming training 2.4. Blood biochemical analyses After 21 days, SL and NL groups were subdivided into exercised (EXE) or sedentary (SED) animals. Swimming training was performed according to the protocol previously described by Gobatto et al [10]. and modified by Leite et al [8]. Briefly, exercised groups swam 3 times/week for 30 minutes for a period of 72 days. Water temperature was maintained at 32 ± 2 C to eliminate cold-induced
After 12 h of fasting other groups of rats (n ¼ 10/group) were killed by decapitation and total blood collected in heparinized tubes. Plasma glucose, cholesterol and triglyceride levels were measured by enzymatic procedures using commercial kits (Gold Analisa®, Belo Horizonte e MG, Brazil) and read by means of an
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automatic analyzer (Selectra II da Bayer). Insulin was measured via RIA. 2.5. Histology Part of the rats (n ¼ 3e4/group) used by biochemical parameters were also used by histological studies. The abdominal cavity of the rats was opened and several tissues were collected for histological analysis according to the procedures described hereafter. The splenic portion (tail) of the whole pancreas was rapidly removed and cleared of fat. The subcutaneous inguinal deposit and the visceral mesenteric and epididymal depots were collected from white adipose tissue. In addition, interscapular brown adipose tissue was also removed. All tissues were fixed in ALFAC solution (85 mL 80% alcohol, 10 mL 40% formaldehyde, and 5 mL glacial acetic acid) for 20e24 h at room temperature. After fixation, tissues were dehydrated in graded ethanol, cleared in xylol, and embedded in paraffin at 60 C. 5 mm sections were cut on the microtome (Leica RM 2025, Germany), stained with hematoxylin and eosin (H&E) and then analyzed with image analysis software. At least 3 slices per adipose tissue sample and 9 fields of view were examined for each slice to determine the adipocyte cross-sectional areas. The cell parameters were measured using an image analysis system (Image J 1.39f, NIH e Bethesda, MD, USA). The sectional areas of the adipocytes and islets were determined from digital images acquired at random (Olympus BX51 microscope, LC Evolution camera, TIFF format, 36-bit color, 1280 1024 pixels). A total of 50 adipocytes were measured per section and all islets were measured in each section. The number of adipocytes and islets was also evaluated. The number of nuclei was evaluated in the brown adipose tissue. Three representative areas were scored in each section, and the average values were used. 2.6. Food and water intake After weaning (21 days), rats from all groups were weighed, and their food and water intake was determined 3 times per week until adult life (93 days). Food and water intake were calculated as the amount consumed divided by the body weight of each animal and are expressed in g/100 g of body weight (bw). In this way, the total area under the curve (AUC) of food and water consumption versus time was calculated. All rats in the cage were of the same group and each rats was individually indentified by tattoo in the tail. 2.7. Obesity Adipose tissue accumulation was evaluated in all experimental groups to confirm obesity in adult life (93 days). Thus, same rats used in protocols described above were studied (n ¼ 10e14/group). The mesenteric and periepididymal deposits were weighed and designated as visceral adipose tissue, while inguinal adipose tissue was weighed and designated as subcutaneous deposits. In addition, interscapular brown adipose tissue was also measured. The weights of adipose tissues and organs were expressed as g/100 g bw. The Lee index [bw (g)1/3/nasal-anal length (cm)] was calculated as a predictor of obesity. During lactation we also evaluated daily the evolution of the body weight of the offspring and at weaning (21 days) the Lee Index of the offspring was also calculated. 2.8. Western blotting assay Isolated islets were pelleted and resuspended in buffer containing protease inhibitors as previously described by Amaral et al. [14]. Total protein content was determined by the Bradford (1976) method [15]. Fifteen (15) mg of total protein extract from pancreatic
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islets were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then incubated with Phospholipase C b (PLC b) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or Muscarinic Receptor 3 (M3) (Sigma Aldrich, St. Louis, Mo., USA) antibodies. Antibody dilutions were made according to the manufacturer's instructions in TRIS-Tween buffer containing 30 g/L of dried skim milk. Band detection was performed by chemiluminescence (Pierce Biotechnology, Rockford, IL) after incubation with a horseradish peroxidase-conjugated secondary antibody. Band intensities were quantified by optical densitometry (Image J, National Institutes of Health, USA). 2.9. Chemical and reagents Analytical grade reagents and deionized water were used. Collagenase type V, BSA (fraction V), acetylcholine and the inhibitor neostigmine were obtained from Sigma (St. Louis, Mo., USA). 2.10. Statistical analysis The results are shown as the mean ± the standard error of the mean (SEM). The data were subjected to Student's t -test or a one way analysis of variance (ANOVA) followed by a Bonferroni posttest analysis with selected pairs grouped when appropriate. P values less than 0.05 (p < 0.05) were considered statistically significant. The statistical tests were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). 3. Results Hypernutrition during lactation induced obesity and hyperglycemia during adulthood in rats according to the results in Table 1. SL-SED rats presented with an increase of 8.7% and 3% in body weight and LI, respectively, when compared to NL-SED animals. In addition, basal glycemia was 16.2% higher in SL-SED rats than in NLSED animals (p < 0.05). The insulin levels, total cholesterol, triglyceride levels and nasal-anal lengths were not significantly different in SL-SED rats. The effects of swimming training in both experimental groups are also represented in Table 1. Both of the exercised groups (NL-EXE and SL-EXE) presented with a reduction of 8.8% and 7.2% in body weight, respectively, when compared to the sedentary groups. Similarly, the LI was decreased by 2.8% and 3.7% in NL-EXE and SL-EXE rats, respectively, when compared to the sedentary groups. In addition, NL-EXE and SL-EXE rats exhibited a reduction of approximately 16% and 20%, respectively, in glycemia, total cholesterol and triglycerides when compared to the NL-SED and SL-SED groups. Fig. 1(A-E) shows body weight, LI, food intake and hydric intake evolution. During lactation (0e21 days), SL rats showed an increase in body weight evolution when compared to NL rats (Fig. 1A). This difference was first observed at postnatal day 12 and persisted for 21 days. Thus, during the lactation phase the AUC of the body weight was increased by 18.5% in SL rats when compared to the NL group (p < 0.05). The LI was also approximately 9.1% higher in SL rats than in NL rats at 21 days (Fig. 1B). Body weight evolution postweaning (22e90 days) is shown in Fig. 1C. SL-SED rats exhibited a slight increase in body weight evolution when compared to NL-SED rats. The AUC of body weight measurements post-weaning was 9.5% higher in SL-SED rats than in NL-SED animals. However, during this period body weight gain was not associated with changes in food or water intake (Fig. 1D and E). In the NL-EXE and SL-EXE groups, swimming training reduced the body weight AUC by 6.7% and 8%, respectively, when compared to the NL-SED and SL-SED rats. In both experimental groups, swimming training did not alter food or water intake.
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Table 1 Effects of swimming training in the biometric and biochemistry parameters in overnourished rats. NL-SED Body weight (g) Lee index Naso-anal length (cm) Basal glucose (mg/dL) Basal insulin (ng/mL) Total cholesterol (mg/dL) Triglycerides (mg/dL)
308.00 0.309 21.77 98.57 0.22 128.40 63.69
± ± ± ± ± ± ±
NL-EXE 3.87b,c 0.002b,c 0.14 4.22b,c 0.03 3.09b 3.90b
282.40 0.300 21.86 83.25 0.18 110.00 51.10
± ± ± ± ± ± ±
SL-SED 3.90a 0.001a 0.14 3.43a 0.03 2.89a 2.68a
335.00 0.318 22.05 114.5 0.29 136.50 64.84
SL-EXE ± ± ± ± ± ± ±
4.41a,d 0.002a,d 0.18 4.27a,d 0.04 8.30d 4.52d
310.70 0.306 22.10 96.35 0.27 100.40 52.45
± ± ± ± ± ± ±
2.90c 0.002c 0.13 5.21c 0.03 2.68c 2.60c
The data are expressed as the mean ± SEM obtained from 15e20 rats by group. Letters above the numbers represent a significant difference of p < 0.05 obtained by analyses of variance ANOVA followed by Bonferroni's posttest (selected par groups). a NL-SED. b NL-EXE. c SL-SED. d SL-EXE.
The impact of hypernutrition and swimming training on adipose tissue content and adipocyte hypertrophy is shown in Fig. 2. The SLSED groups exhibited increases in white visceral adipose tissue when compared to NL-SED rats. The mesenteric (Fig. 1-L) and retroperitoneal (Figure E-H) content were approximately 25%
higher in SL-SED rats than in NL-SED groups. However, subcutaneous inguinal fat and brown adipose tissue content were similar between SL-SED and NL-SED rats. The visceral white adipose tissue content was reduced by approximately 20% in NL-EXE rats when compared to the NL-SED group. Similarly, swimming training
Fig. 1. Effects of swimming training on body weight and food and water intake of overnourished rats. Values are expressed as the mean ± SEM of 15e20 rats for all groups. Body weight and ìndice Lee was evaluated during lactation (1 at 21 days, Fig. 1A and B respectively) and after weaning (22 at 90 days, Fig. 1C). The food and water intake are represented in the lower panels (Fig. 1D and E). The respective areas under the curve (AUC) are shown in the insets. * represents a significant difference of p < 0.05 between the NL and SL groups obtained by Student's t test. Letters above the bars show significant differences of p < 0.05 obtained by an ANOVA followed by Bonferroni's posttest (selected par groups). *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE.
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Fig. 2. White and Brown adipose tissue content and morphological aspects of SL-exercised rats. Representative photomicrographs of white visceral adipocytes (Fig. 2A-L), subcutaneous adipocytes (Fig. 2M-P) and brown adipocytes (Fig. 2Q-T). The photomicrographs were assessed by H&E staining (200 and 400, white and brown adipocytes, respectively) with scale bars indicating 50 mm and 20 mm. The diameter and number of white adipocytes were evaluated. In brown adipocytes, proliferation was evaluated by the number of nuclei. Respective differences between groups are described in the results section. The lower figures (2U-2Y) represent white and brown adipose tissue weights. Values are expressed as the mean ± SEM of 15e20 rats for all groups. Letters above numbers show significant differences of p < 0.05 obtained by an ANOVA followed by Bonferroni's posttest (selected par groups). *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE.
reduced white visceral adipose tissue content by approximately 19% in SL-EXE animals when compared to SL-SED rats (p < 0.05). In both groups, subcutaneous inguinal fat (Figure M-P) content was not affected by swimming training. In contrast, brown adipose tissue content increased 106.8% and 63.9% for the NL-EXE and SLEXE groups, respectively, when compared to their sedentary counterparts (p < 0.05). Hypernutrition during lactation induces hypertrophy in white adipocyte tissue according to the morphological data presented in Fig. 2 and Table 3. Adipocytes isolated from epididymal, retroperitoneal and inguinal fat contained approximately 20% more adipocytes in SL-SED rats when compared to NL-SED animals (p < 0.05). Adipocyte areas from mesenteric fat were similar between SL-SED and NL-SED rats. Only epididymal fat content exhibited a reduction of 15% in adipocyte number in SL-SED rats when compared to NL-SED animals. Similarly, the number of adipocytes in brown adipose tissue exhibited a reduction of 25.4% in the SL-SED group when compared to NL-SED rats (p < 0.05). Swimming training reduced the area of the adipocytes by approximately 16% in epididymal and retroperitoneal fat, 23.3% in mesenteric fat and 8.6% in inguinal fat in NL-EXE groups when compared to NL-SED animals. Similarly, in SL-EXE rats the area of the adipocytes was reduced by approximately 18.5% in epididymal
deposits, 32.4% in retroperitoneal deposits and 13.6% in inguinal fat deposits when compared to adipocytes isolated from SL-SED rats (p < 0.05). Glucose-induced insulin secretion and pancreatic islet morphology in SL rats subjected to swimming training are shown in Fig. 3. Pancreatic islets from SL-SED rats presented with a reduction in glucose-induced insulin secretion. In the presence of high glucose concentrations, pancreatic islets isolated from SL-SED rats exhibited a reduction in glucose-induced insulin secretion of approximately 33% when compared to islets obtained from the NLSED groups. Swimming training caused a reduction in glucoseinduced insulin secretion in pancreatic islets from NL-EXE rats when compared to islets isolated from the NL-SED group. In contrast, pancreatic islets from SL-EXE rats exhibited an increase in glucose-induced insulin secretion of approximately 95% when compared to islets isolated from SL-SED animals (p < 0.05). Pancreatic islets isolated from SL-SED animals exhibited reductions in area and number of 34.2% and 30%, respectively, when compared to islets isolated from NL-SED rats (p < 0.05). In both groups, swimming training altered the amount of proliferation and the size of the pancreatic islets. The NL-EXE and SL-EXE groups exhibited an increase in pancreatic islet number of 31.7% and 39.6%, respectively, when compared to their sedentary counterparts. In addition, islet
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Fig. 3. Glucose-induced insulin secretion and histological analysis of pancreatic islets isolated from SL-exercised rats. Pancreatic islets were isolated by the collagenase technique as described in the methods section and incubated in the presence of different glucose concentrations (Fig. 3A). Values are expressed as the mean ± SEM of 20e24 observations for each glucose condition. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SLEXE. Fig. 3B shows representative photomicrographs of pancreatic islets assessed by H&E staining (400) with scale bars indicating 200 mm. The diameter and number of pancreatic islets were evaluated. Individual islet profiles are shown in the insets. Respective data are described in the results section.
area was reduced by 41.1% and 39.3% in the NL-EXE and SL-EXE groups, respectively, when compared to islets isolated from the sedentary groups (p < 0.05). The effects of hypernutrition and swimming training on cholinergic-induced potentiation and islet protein expression were also evaluated (Fig. 4A-C). The addition of Ach resulted in a dosedependent increase in insulin secretion from islets isolated from
Fig. 4. Cholinergic insulinotropic response of islets isolated from overnourished rats subjected to swimming training (A) and expression of M3 (B) and PLC b (C). Groups of 4 islets were incubated in 1 mL of Krebs solution with glucose (8.3 mM) in the presence of different concentrations of acetylcholine. The data are shown relative to 100% (only glucose concentration is 8.3 mM). Four or six rats were sacrificed from each group to form a pool of islets. Values are expressed as the mean ± SEM of 20e24 observations for each acetylcholine condition. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE. Representative western blotting images are shown and densitometry quantification is indicated by vertical bars for M3 and PLC b for all groups. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. DNL-SED versus NL-EXE.
the NL-SED group. In these islets, high Ach concentrations (such as 100 and 1000 mM) increased insulin secretion by approximately 6 fold (p < 0.05). Pancreatic islets isolated from the NL-EXE group exhibited an attenuated Ach insulinotropic effect, which was approximately 50% less than that observed in islets isolated from NL-SED rats. Hypernutrition during lactation altered the cholinergic-induced potentiation of pancreatic islets. In the presence of low or moderate Ach concentrations (0.1e10 mM) the cholinergic potentiation effect was inhibited (by approximately 104%) in islets isolated from the SL-SED group, whereas cholinergic-induced effects were observed only in the presence of the highest doses of Ach (100e1000 mM). Swimming training accentuated the inhibitory response of Ach in islets isolated from the SL-EXE group (p < 0.05). Muscarinic receptor (M3) and PLCb expression of the isolated pancreatic islets are represented in Fig. 4B and C, respectively. There was a reduction in M3 expression in the NL-EXE group (of 44.8%) when compared to NL-SED rats (p < 0.05). The expression of PLCb was not affected by hypernutrition or exercise training. The insulinotropic effects of GLP1 on glucose-induced insulin secretion are shown in Fig. 5(A and B). In the presence of low
Fig. 5. GLP-1 stimulated action of glucose-induced insulin secretion in islets isolated from overnourished rats subjected to swimming training. Groups of 4 islets were incubated in 1 mL of Krebs solution with 5.6 mM glucose (Fig. 5A) or 16.7 mM glucose (Fig. 5B) in the presence of GLP-1 (10 nM). The data are shown relative to 100% (5.6 or 16.7 mM glucose only controls). Four or six rats were sacrificed from each group to form a pool of islets. Values are expressed as the mean ± SEM of 20e24 observations for each glucose condition. Symbols above the bars represent a difference of p < 0.05 obtained by Student's t test. *NL-SED versus SL-SED; #NL-SED versus NL-EXE; &SL-SED versus SL-EXE.
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glucose (5.6 mM), insulin secretion increased by 73% and 158% in islets isolated from NL-SED and SL-SED rats, respectively. Under these glucose conditions, both exercised groups exhibited a reduction in the GLP1 insulinotropic effect (Fig. 5A, p < 0.05). However, in the presence of a high glucose concentration (16.7 mM), GLP1-stimulated action was observed only in islets isolated from SL rats. The insulinotropic effect of GLP1 was 260% and 115% in islets isolated from SL-SED and SL-EXE rats, respectively (Fig. 5B, p < 0.05). 4. Discussion Reduction in litter size during the suckling period can induce nutritional programming that alters the metabolism of offspring during adult life [2]. Postnatal suckling period is a vulnerable period from offspring because in this phase many organs, as well as, important energetic hypothalamic pathways are established [16]. Consistent with these reports, in our study pups assigned to small litter showed an increase in body weight gain and LI during suckling period remaining overweight at 93-old-days. Similarly in humans, several observations have shown that rapid weight gain in the early stages predispose at overweight as well as the future development of obesity in adulthood [17]. Our study also demonstrated that in SL-adult rats the increase in body weight gain was accomplished of rises in fat body content and fasting hyperglycemia. It has been demonstrated that reduction in the litter size during lactation promotes overweight, glucose tolerance and hyperphagia in adulthood [2,18]. However, in our study was not observed hyperphagia in SL-adult rats. The reason behind these inconsistent results could involve differences in food intake expression according previously established by Xiao et al [19]. In our study we also did not observe dyslipidemia or hyperinsulinemia in SL-adult rats, corroborating with results obtained by Rodrigues et al. with SL-rats of the same age [18]. Independently these divergent data, in our study the obesity in SL-adult rats can be confirmed by high white adipose tissue content accompanied by adipocyte hypertrophy. In addition, we also demonstrated that hypernutrition during lactation increase the size and number of lipid droplets in brown adipose tissue from SL-adult rats, suggesting reduction in brown adipose tissue thermogenesis. This confirm results obtained by Xiao et al. in SL-rats [19]. Our swimming training was started at 21 days, in this age SLrats already presented heavier body weight and increased LI. Chronic swimming training effectively avoided obesity, hiperglycaemia, reduced lipid levels and adipose tissue content in SLtrained rats. Thus, our study demonstrated by the first time that SL-rats are responsive at benefits of the aerobic exercise program started immediately after weaning and maintained until adult life. These data also corroborated with study realized by Scomparin et al. showing that precocity of the exercise training is important to avoid obesity abnormalities [12]. The development of the endocrine pancreas is vulnerable to nutritional insults during the immediate post-natal phase [1]. Pancreatic endocrine dysfunction is associated with a reduction in beta cell mass and impaired glucose-stimulated insulin secretion [4]. In our study, glucose-induced insulin secretion and islets number were reduced in endocrine pancreas from SL-adult rats. Similar results were obtained by Waterland and Garza. [1]. According with these authors the endocrine pancreas is the primary tissue affected by metabolic imprinting. The central aspect of our study was to evaluate the influence of swimming training on the endocrine pancreas of SL-adult rats. Interestingly, we observed that swimming training was able to restore glucose responsiveness in islets isolated from SL-adult rats. A study performed by Dela et al. showed that physical training
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increased glucose-induced insulin secretion in pancreatic islets isolated from diabetic patients [6]. According to the authors, this pancreatic response to exercise is directly dependent upon beta cell secretory capacity and diverges in health and diabetic conditions. In addition, we also demonstrated that swimming training promoted increases in islet numbers and a simultaneous reduction of islet size in pancreas samples isolated from SL-adult rats. Shima et al. showed that physical training improves islet morphological alterations observed in Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats with T2D [11]. Similarly, Park et al. demonstrated that exercise can stimulate pathways of proliferation in islets of diabetic rats [20]. Taken together, these results indicate that swimming training started at the post-weaning stage and maintained during development can avoid abnormalities in glucose responsiveness induced by hypernutrition during suckling. Disruption in cholinergic insulinotropic action is directly involved in pancreatic islet dysfunction [21,22]. Cholinergic effects on pancreatic beta cells are exerted primarily through muscarinic receptors (MRs). The presence of multiple MRs (1e4) has been demonstrated in pancreatic islets or beta cells [23], however MR subtype 3 (MR3) appears to be the predominant subtype expressed by pancreatic beta cells [22]. For the first time, our study evaluated the Ach response of pancreatic islets isolated from overnourished rats. A cholinergic inhibitory response was observed in islets isolated from SL-adult rats at low concentrations of Ach, whereas a weak insulinotropic effect was observed at high concentrations of Ach. Similarly, pancreatic islets isolated from undernourished rats (a model of metabolic programming) exhibited a reduction in MR3 activation [24]. Moreover, studies have also demonstrated that Ach can inhibit glucose-induced insulin secretion via MR subtype 2 or 4 (MR2 or 4) [23,25]. Thus, we suggest that altered composition of MR may be involved in these abnormal cholinergic responses in pancreatic islets isolated from SL-rats because MR3 or PLCb expression did not change in this group of islets. This possibility was recently confirmed by de Oliveira et al. in islets isolated from undernourished rats [26]. Surprisingly, our results also demonstrate that swimming training accentuated the inhibitory action of Ach on islets obtained from both groups. In islets obtained from lean rats, these responses seem to involve a reduction in MR3 expression. Contrary data were obtained by Almeida et al. In this study, lean trained animals submitted to an acute exercise regimen increased their insulin secretion stimulated by the cholinergic agent carbachol [7]. Divergent achieves could be attributed at acute or chronic effect of the exercise training. Thus, we suggested that weak cholinergic insulinotropic effect in islets from exercised rats could be the result of an adaptation to avoid hypoglycemia once that physical training also improves insulin sensitivity. Finally, we also observed that islets from SL-adult rats exhibit increased GLP1 insulinotropic action. GLP-1 can promote glucosestimulated insulin secretion and stimulates pancreatic beta cell masses [27]. This incretin hormone is primarily produced by intestinal L cells; however, recent evidence indicates that GLP1 may also be produced in the islets [28]. There are no known studies that evaluate GLP1 action or production in SL-overnourished rats. However, high-carbohydrate (HC) milk formula offered during the suckling period can also induce hypernutrition [5]. Similar to our results, islets isolated from HC rats exhibit increased insulinotropic GLP1 responses. In addition, Miguel et al. have demonstrated the cooperative insulinotropic effects of Ach and GLP1 in a beta tumor cell line [29]. Thus, we suggest that the high GLP1 insulinotropic action observed in islets isolated from overnourished rats could be provoked by altered GLP1 expression in pancreatic islets and/or by the altered cholinergic effects demonstrated by our results. Our study also demonstrates that swimming training reduced GLP1 insulinotropic action at low glucose concentrations. Similar results
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were obtained by Svidnicki et al. in pancreatic islets isolated from hypothalamic obese rats subjected to swimming training [30]. These results seem to indicate that physical training can correct GLP1 insulinotropic action in pancreatic beta cells isolated from overnourished rats. This study demonstrates that early swimming training can prevent obesity installation, correct glucose-induced insulin secretion, as well as, GLP1 insulinotropic response in pancreatic islets isolated from overnourished rats without altering the abnormal cholinergic response. Strengths and Limitations: Our research shows clearly that the exercise introduced early during development is capable of avoids the deleterious effects induced by metabolic program. This aspect is of the particular interest to blockage obesity progression among children, once that, research into the 'developmental origins of health and disease' (DOHaD) has now firmly established that preand perinatal developmental perturbations can predispose to obesity in adult life. However, our study also presents limitations. The effect of metabolic program is sex-dependent, our study evaluated only male rats, thus we cannot affirm that similar effects would be observed in female. In addition, more adequate technique is necessary to evaluate exercise program (time, intensity and frequency) before of extrapolates to early childhood development. Statement of authorship Sabrina Grassiolli and Stefani Valeria Fisher designed the udia Regina Capriglioni Cancian, research. Stefani Valeria Fisher, Cla and Elisangela Gueiber Monte, conducted the research and analyzed the data. Nayara de Carvalho Leite performed the molecular techniques. Stefani Valeria Fisher and Sabrina Grassiolli wrote the paper. Stefani Valeria Fisher assumes primary responsible for this manuscript's final content. All authors read and approved the final manuscript. Conflict of interest statement and funding sources None of the authors declare a conflict of interest. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. We are grateful to Coor~o de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) denaça for the scholarship offered to Post-Graduate Student Stefani Valeria Fisher. Acknowledgments The authors would like to thank Professor Paulo Cesar de Freitas Mathias and Julio Cesar de Oliveira from Universidade Estadual de (UEM) e Parana e Brasil for assistance with the radioimMaringa ~es Carneiro munoassay technique and Professor Everardo Magalha from Universidade Estadual de Campinas e Campinas e Brasil for molecular technique support. References [1] Waterland RA, Garza C. Early postnatal nutrition determines adult pancreatic glucose-responsive insulin secretion and islet gene expression in rats. J Nutr 2002;132(3):357e64. €tz F, Rohde W, Do €rner G. Obesity and enhanced [2] Plagemann A, Heidrich I, Go diabetes and cardiovascular risk in adult rats due to early postnatal overfeeding. Experimental and Clinical Endocrinology & Diabetes 1992;99(03):154e8. [3] Cottrell EC, Ozanne SE. Developmental programming of energy balance and the metabolic syndrome. Proc Nutr Soc 2007;66(2):198e206. [4] Weir GC, Bonner-Weir S. Five stages of evolving beta-cell dysfunction during progression to diabetes. Diabetes 2004;53(Suppl. 3):S16e21. [5] Srinivasan M, Aalinkeel R, Song F, Patel MS. Programming of islet functions in the progeny of hyperinsulinemic/obese rats. Diabetes 2003;52(4):984e90.
[6] Dela F, Michael E, Mikines KJ, Galbo H. Physical training may enhance b-cell function in type 2 diabetes. Am J Physiol Endocrinol Metab 2004;287(5): E1024e31. [7] Almeida FN, Proença AR, Chimin P, Marçal AC, Bessa-Lima F, Carvalho CR. Physical exercise and pancreatic islets: acute and chronic actions on insulin secretion. Islets 2012;4(4):296e301. [8] Leite NC, Ferreira TR, Rickli S, Borck PC, De Freitas Mathias PC, De Oliveira Emilio HR, et al. Glycolytic and mitochondrial metabolism in pancreatic islets from MSG-treated obese rats subjected to swimming training. Cell Physiol Biochem 2013;31(2e3):242e56. cary S, Peyot M-L, Latour MG, Lamontagne J, [9] Delghingaro-Augusto V, De Paradis-Isler N, et al. Voluntary running exercise prevents b-cell failure in susceptible islets of the Zucker diabetic fatty rat. Am J Physiol Endocrinol Metab 2012;302(2):E254e64. [10] Gobatto CA, Mello MaR De, Sibuya CY, De Azevedo JRM, Dos Santos LA, Kokubun E. Maximal lactate steady state in rats submitted to swimming exercise. Comp Biochem Physiol A Mol Integr Physiol 2001;130(1):21e7. [11] Shima K, Shi K, Sano T, Iwami T, Mizuno A, Noma Y. Is exercise training effective in preventing diabetes mellitus in the Otsuka-Long-EvansTokushima fatty rat, a model of spontaneous non-insulin-dependent diabetes mellitus? Metabolism 1993;42(8):971e7. [12] Scomparin D, Grassiolli S, Marçal A, Gravena C, Andreazzi A, Mathias P. Swim training applied at early age is critical to adrenal medulla catecholamine content and to attenuate monosodium L-glutamate-obesity onset in mice. Life Sci 2006;79(22):2151e6. [13] Lacy PE, Kostianovsky M. Method for the isolation of intact islets of langerhans from the rat pancreas. Diabetes 1967;16(1):35e9. [14] Amaral M, Ueno M, Carvalheira J, Carneiro E, Velloso L, Saad M, et al. Prolactinsignal transduction in neonatal rat pancreatic islets and interaction with the insulin-signaling pathway. 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[24] Batista TM, Ribeiro RA, Amaral AG, De Oliveira CA, Boschero AC, Carneiro EM. Taurine supplementation restores glucose and carbachol-induced insulin secretion in islets from low-protein diet rats: involvement of Ach-M3R, Synt 1 and SNAP-25 proteins. J Nutr Biochem 2012;23(3):306e12. [25] Grassiolli S, Gravena C, De Freitas Mathias PC. Muscarinic M2 receptor is active on pancreatic islets from hypothalamic obese rat. Eur J Pharmacol 2007;556(1):223e8. [26] Oliveira J, Scomparin D, Andreazzi A, Branco R, Martins A, Gravena C, et al. Metabolic imprinting by maternal protein malnourishment impairs vagal activity in adult rats. J Neuroendocrinol 2011;23(2):148e57. [27] Drucker DJ. Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care 2003;26(10):2929e40. [28] Marchetti P, Lupi R, Bugliani M, Kirkpatrick C, Sebastiani G, Grieco F, et al. A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets. Diabetologia 2012;55(12):3262e72. [29] Miguel JC, Abdel-Wahab YH, Green BD, Mathias PC, Flatt PR. Cooperative enhancement of insulinotropic action of GLP-1 by acetylcholine uncovers paradoxical inhibitory effect of beta cell muscarinic receptor activation on adenylate cyclase activity. Biochem Pharmacol 2003;65(2):283e92. [30] Svidnicki PV, Leite NDC, Venturelli AC, Camargo RL, Vicari MR, Almeida MC, et al. Swim training restores glucagon-like peptide-1 insulinotropic action in pancreatic islets from monosodiumglutamate-obese rats. Acta Physiol 209(1) 2013:34e44.