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Effects of cafeteria diet on the jejunum in sedentary and physically trained rats
Nutrition 26 (2010) 312–320
Basic nutritional investigation
www.nutritionjrnl.com
Effects of cafeteria diet on the jejunum in sedentary and physically trained rats Ce´lia Regina Scoaris, M.D.a,*, Gabriela Vasconcelos Rizo, M.D.a, Luciana Patrı´cia Roldi, M.D.a, Solange Marta Franzo´i de Moraes, Ph.D.a, Andre´ Ricardo Gomes de Proenc¸a, M.Sc.a, Rosane Marina Peralta, Ph.D.b, and Maria Raquel Marc¸al Natali, Ph.D.a a
Department of Morphophysiological Sciences, State University of Maringa´, Maringa´, Parana´, Brazil b Department of Biochemistry, State University of Maringa´, Maringa´, Parana´, Brazil Manuscript received December 4, 2008; accepted April 15, 2009.
Abstract
Objective: The effects of a cafeteria diet on the small intestine were investigated in adult Wistar rats under sedentary conditions and after physical training. Methods: Parameters including morphometry, enzyme activities, and total myenteric populations in the jejunum were evaluated. Results: The cafeteria diet, characterized as hyperlipidic, produced obese rats, corroborated by increases in the Lee index and the weights of the periepididymal and retroperitoneal adipose tissues (P < 0.01). Obesity caused increases in the length of the small intestine, villi height, crypt depth, whole-wall thickness (P < 0.05), and the enzymatic activities of alkaline phosphatase, lipase, and sucrase (P < 0.01), in addition to a reduction in the number of goblet cells (P < 0.05). With reference to the jejunal intrinsic innervations, the total number and area of myenteric neurons was unchanged regardless of the group. Physical training promoted 1) a reduction of the weight in the retroperitoneal and periepididymal adipose tissues (P < 0.05) and 2) an increase in the thickness of the muscular layer (P < 0.05). Conclusion: The cafeteria diet promoted obesity in rodents, leading to alterations in morphometry and enzymatic intestinal parameters, which were partily attenuated by physical training. Ó 2010 Elsevier Inc. All rights reserved.
Keywords:
Cafeteria diet; Intestinal morphometry; Jejunum; Myenteric neurons; Obesity; Physical training
Introduction The increasing incidence of obesity is a serious public health issue worldwide. According to the World Health Organization, there are more than 1 billion overweight adults with a body mass index (BMI) 25 kg/m2 and around 300 million people with a BMI 30 kg/m2 [1]. The etiology of obesity is extremely complex because of the involvement of neurologic, endocrine, genetic, and behavioral factors. Obesity is characterized by increased energy storage and This work was financed by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico and Fundac¸a˜o Arauca´ria. *Corresponding author. Tel.: þ55-44-3261-4704; fax: þ55-44-32228866. E-mail address: [email protected] (C. R. Scoaris). 0899-9007/10/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.04.012
hypertrophic and hyperplastic alterations in adipocytes, leading to weight gain from an energy imbalance [2]. Health complications attributable to obesity frequently described in the literature include diabetes, cardiovascular disease, orthopedic and postural alterations, breathing and emotional disorders, hepatic steatosis, and gastrointestinal alterations, which affect the quality of life and increase the risk of death. The current obesity epidemic requires a change in life habits, considered from an evolutionary point of view. Previously, men were adapted to periods of physical effort for survival because of a lack of food and the need to obtain fresh food and fiber. At present, humanity leads a more sedentary life, associated with industrialized, refined aliments, saturated fat, and low fiber. The impact of obesity can be decreased through regular physical activity, restoration of
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a positive energy balance, promotion of antioxidant defense mechanisms, and by decreasing the occurrence of diseases associated with oxidative stress [3]. Experimental animal models resembling human obesity include those in which a hyperlipidic diet (also referred to as a cafeteria diet), Western diet, or fast-food diet is used [4–8]. This diet, although with increased energy value, has low nutritive value, because it is overloaded with carbohydrates, fat, or both [4]. This type of diet is characterized by its capacity to induce not only increased lipid storage in the adipose tissue but also increased oxidative stress throughout the system [5]. The small intestine is responsible for digesting and absorbing nutrients and is structurally adapted to carry out these functions. It has a large surface area and functions in triggering signals to the central nervous system and ensuring energy homeostasis [9]. The intestinal mucosa is sensitive to alterations in this environment, with hypertrophy when there is food overload and atrophy when there is a lack of food [10]. In obesity conditions induced by the administration of monosodium glutamate (MSG), intestinal morphometric parameters, such as the villi, crypts, and muscular-coat thickness, were maintained in the jejunum of mice [11] and ileum of rats [12]. Enzymatic activity can be altered according to the alimentary substrate. Increased activity of alkaline phosphatase was found in the jejunum of obese rats due to the intake of a hyperlipidic diet [13]. The small intestine is extrinsically innervated by the autonomic nervous system, and its activity is modulated intrinsically by the enteric nervous system. This system is organized into a complex network of nerve fibers and neuronal cell bodies (e.g., interneurons, motor and sensory neurons). The myenteric plexus is one of the main ganglionated plexuses comprising the enteric nervous system, which is located between the inner circular muscular coat and the outer longitudinal muscular coat of the digestive tract and responds to intestinal motility, among other functions [9,12,14–19]. Morphologic and quantitative variations in the enteric neurons occur according to age [14,15], denervation model [16], diabetes [17], diet restriction [18], obesity induced by neonatal administration of MSG [12], and physical training [19]. Because myenteric neurons are sensitive to alterations in the tension of the intestinal wall and the chemical environment of the intestine, the present study aimed to evaluate morphometric and enzymatic parameters, in addition to the population of myenteric neurons, in the jejunum of obese rats, which were sedentary or subjected to physical training. Materials and methods Animals The present study used male 70-d-old Wistar rats (Rattus norvegicus) with an initial average weight of 270 g from the Animal House of the State University of Maringa´. All animal
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procedures of the study were approved by the committee of ethics on animal experimentation of the State University of Maringa´. According to the diet type and physical activity, 28 animals were allocated to four groups: sedentary normally fed rats (SN), sedentary rats fed a cafeteria diet (SCa), trained normally fed rats (TN), and trained rats fed a cafeteria diet (TCa). During the experimental period of 120 d, animals were housed in polypropylene boxes at room temperature (24 6 2 C) under a 12-h light/12-h dark cycle. Diet Animals from the SN and TN groups received standard rodent chow (Nuvilab-Nuvital (Curitiba, PR, Brazil), recommended by the National Research Council and National Institutes of Health, Bethesda, MD, USA) and water ad libitum. Animals from the cafeteria groups (SCa and TCa) received a cafeteria diet [4–8] consisting of the usual pellets of standard diet, cheese or bacon-flavored chips, bread, chocolate, marshmallows, peanut candy, filled cookies, wafer cookies, sausage, and mortadella, in addition to ad libitum soda and water. The standard rodent diet and cafeteria diet were analyzed in terms of the percentage of macronutrients and fiber content by the Laboratory of Animal Nutrition, Zootechny Department, Maringa´ State University. Diet consumption, in grams and calories, was measured daily, and liquid consumption and body weight were measured three and two times per week, respectively. Physical training After 30 d of feeding on a cafeteria diet, animals were subjected to physical training for a period of 12 wk, consisting of treadmill running (Inbrasport, Porto Alegre, Brazil) from 16:30 to 18:30 h, with a previous training adaptation period of 10 min, at speeds ranging from 0.3 to 0.6 km/h. The training protocol [20] consisted of running five times per week, ranging from 15 to 60 min of training per session, with a variable speed from 0.3 to 1.4 km/h and no change of treadmill tilt. Tissue collection When animals were 190 d old, they were weighed, anesthetized with sodium thiopental (Thionembutal (Sa˜o Paulo, Brazil), 40 mg/kg, intraperitoneally), and their nasal–anal length was measured to obtain the Lee index (body weight1/3 [g]/nasal–anal length [cm] 3 1000) [21]. Subsequently, laparotomy was carried out and the periepididymal and retroperitoneal adipose tissues were dissected out from the small intestine; the weights and lengths (from the pylori to the ileocecal junction) of these dissected samples were measured. The jejunum was isolated, its width was measured, and it was divided into samples, which were further subjected to the following protocols: histologic processing and
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embedding in paraffin for the morphometric study of the muscular coat and total wall; inclusion in historesin for the morphometric evaluation of intestinal crypts and villi; histochemistry to evaluate goblet cell population; analysis of intestinal enzymes (alkaline phosphatase, lipase, b-galactosidase, maltase, and sucrase); and whole-mount preparations to evaluate the total myenteric population. Morphometric analysis of the jejunal wall Jejunal samples of five animals from each group were collected, opened along the mesenteric insertion, washed with saline solution, and adhered in polystyrene. Samples were fixed in Bouin’s solution for 6 h, dehydrated using gradedconcentration solutions of ethanol (70%, 80%, 90%, and 100%), diaphanized in xylol, and embedded in histologic paraffin. Transverse semi-serial sections of 5-mm thickness were obtained using a Leica RM 2145 microtome (Leica Microsystems, Wetzlar, Germany) with a steel knife. The histologic sections were stained with hematoxylin and eosin to evaluate the muscular coat and intestinal wall (from the top of the villi to the serosa coat). Subsequently, 50 points each (micrometers) were measured at random in the muscular coat and intestinal wall per animal. Morphometric analysis of intestinal villi, crypts, and goblet cell populations To evaluate villi height, crypt depth, and total population of goblet cells, the jejunum samples of five animals per group were collected and opened along the mesenteric insertion and fixed in Bouin’s solution. After fixation, they were dehydrated and embedded in 2-hydroxy-methacrylate resin (Leica Microsystems). Transverse semi-serial sections of 2-mm thickness were obtained using a Leica RM 2145 microtome; the sections were stained with hematoxylin and eosin to measure the height of 90 villi and the depth of 90 crypts per animal. Semi-serial sections were also subjected to the histochemical periodic acid–Schiff staining technique to quantify the goblet cell population in 50 microscopic fields (0.352 mm2) per animal from 10 histologic sections. Morphometric analysis was carried out using the images obtained from a highresolution camera (Q Color 3 Olympus American, Burnaby, BC, Canada) coupled to an Olympus BX 41 microscope (Olympus, Tokyo, Japan); subsequently, these images were transmitted to a computer using Q Capture Pro 5.1 and Image-Pro Plus 4.5 (Media Cybernetics, Silver Springs, MD, USA). Intestinal enzyme levels Jejunal samples of five animals from each group were collected, frozen in liquid nitrogen, and stored in a freezer at 80 C. Samples were weighed, cut out, macerated with treated sand, suspended in 4 mL of sodium phosphate buffer
(50 mM, pH 6.5), and centrifuged for 15 min at 4 C at 4000 rpm. The supernatant was further used to determine the levels of alkaline phosphatase [22], lipase [23], sucrase [24], b-galactosidase [22], and maltase [25]. For all enzymes, a unit of enzymatic activity was defined as the enzyme quantity that produced 1.0 mmol of product per milliliter within 1 h under experimental conditions. The specific activity was expressed as units per gram of jejunum (wet weight). Whole-mount preparations Jejunal samples of seven rats from each experimental group subjected to whole-mount preparations were used for this study. The samples were stained by the Giemsa method [26] that allows estimations of the total myenteric population. Samples were excised, and the lumen, after washing with saline solution, was filled with Giemsa fixer using a syringe. The edges were tied to form a ‘‘balloon’’ and immersed in the same solution for a period of 24 h. Subsequently, the samples were opened along their mesenteric border and microdissected under a transillumination stereomicroscope, removing the mucosa and submucosa coats, while simultaneously preserving the muscular and serosa coats, to obtain the whole-mount preparations. These samples were stained with Giemsa stain, consisting mainly of methylene blue in 0.1 N Sorensen buffer (pH 6.9), under agitation for 24 h. The samples were then dehydrated in alcohol, diaphanized in xylol, assembled over slides, and coverslipped with Permount synthetic resin. Quantitative analysis and neuronal morphometry The myenteric neurons present in 80 microscopic fields were randomly quantified using an optical microscope (Olympus) with a 403 objective for the whole-mount preparations in the intermediate (60–120 and 240–300 degrees) and antimesenteric (120–240 degrees) regions, with reference to the mesenteric insertion of the intestinal circumference [27]. Each field corresponded to 0.224 mm2, totaling 17.92 mm2/animal. The measurements (square micrometers) of the cell bodies of 100 neurons/animal, for a total of 700 neurons per studied group, were obtained using computerized image analysis (Image Pro Plus 4.5, Media Cybernetics). The mean value 6 standard deviation for each group was obtained, and the neurons were distributed in class intervals of 100 mm2 according to neuronal area. Statistical analysis Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA). Two-way analysis of variance was followed by Bonferroni’s post hoc test to compare the mean values. Levels of p < 0.05 were considered statistically significant. Results are presented as mean 6 standard deviation.
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Table 1 Alimentary consumption, caloric consumption, percentages of protein, lipid, and fiber; and consumption of daily water, soda, and total liquid in SN, SCa, TN, and TCa rats*
Consumption (g) Consumption (kcal) Protein (%)y Lipids (%)y Fiber (%)y Water (mL) Soda (mL) Total liquid (mL)
SN
SCa
TN
TCa
25.92 6 2.52 101.10 6 8.83 22.23 4.00 5.73 40.19 6 10.55 — 40.19 6 10.55
25.32 6 4.41 89.63 6 14.63 9.98 16.22 1.46 14.98 6 7.09 24.62 6 13.40 39.60 6 16.69
24.41 6 1.79 95.24 6 7.01 22.23 4.00 5.73 39.90 6 9.87 — 39.9 6 9.87
26.65 6 5.02 95.48 6 15.88 9.98 16.22 1.46 13.72 6 6.57 27.22 6 13.11 40.94 6 14.65
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 7 per group). y Laboratory of Animal Nutrition, Zootechny Department, Maringa´ State University.
and thereby produced obese rats. Physical training did not attenuate this condition. The highest weight in the adipose tissues (periepididymal and retroperitoneal), in addition to their sum, was found in the SCa and TCa groups compared with the other groups. Obesity was confirmed in these rodents. The weights of the periepididymal and retroperitoneal adipose tissues, and their sum, were reduced in trained animals regardless of their nutritional condition.
Results Alimentary consumption and macronutrient percentage The results refer to alimentary, caloric, and liquid consumption, in addition to percentages of the macronutrients and fiber content of the diets (Table 1). The cafeteria diet is characterized as hyperlipidic, hypoproteic, and isocaloric, associated with reduced fiber content. The lack of significant differences in the alimentary intake in all groups indicates that diet and physical training did not alter the alimentary intake of these animals and that they were, hence, normophagic. Total liquid consumption was similar among all the groups studied; however, consumption of soda by the SCa and TCa groups was higher compared with that of water.
Intestinal morphometry The values obtained for intestinal length in the SN, SCa, TN, and TCa groups, respectively, were 118.86 6 6.04, 124.43 6 5.26, 121.57 6 3.46, and 124.43 6 3.26 cm. The values obtained for intestinal width were 1.01 6 0.38, 0.87 6 0.4, 0.80 6 0.18, and 0.86 6 0.15 cm, respectively. The lengths of the small intestine were significantly different in the groups that received the cafeteria diet. The morphometric results regarding villi height, crypt depth, intestinal total wall, and muscular-coat thickness and the quantification of total goblet cell population are presented in Table 3. The cafeteria diet increased the villi height, crypt depth, and total wall thickness. Physical training did not influence these parameters. Physical training promoted a significant increase in the muscular coat in animals of the physical training groups compared with the control group.
Validation of the obesity model Results validating the induction of the obesity condition subsequent to consumption of a cafeteria diet are presented in Table 2. Animals that consumed this diet for 120 d gained more weight compared with normally fed animals. Physical training was not effective in reducing or controlling weight gain. Trained TN and TCa groups were not different in terms of weight from the SN and SCa groups. The Lee index, a parameter considered analogous to the BMI, was high in animals that received the cafeteria diet Table 2 FW, Lee index, PER and RET weights, and S in SN, SCa, TN, and TCa rats* SN FW (g) Lee indexy PER (g/100 g body weight) RET (g/100 g body weight) S (g)
422.30 6 39.50 304.60 6 7.40 1.29 6 0.40 1.48 6 0.34 2.77 6 0.70
SCa z
523.20 6 52.00 322.20 6 11.30z 2.30 6 0.52z 3.54 6 0.64z 5.84 6 0.82z
TN
TCa
393.90 6 37.70 303.90 6 5.40 0.89 6 0.13x 1.09 6 0.24x 1.98 6 0.34x
522.80 6 47.50z 320.10 6 7.00z 2.01 6 0.35zx 3.23 6 0.41zx 5.24 6 0.70zx
FW, final body weight; PER, weight of periepididymal adipose tissue; RET, weight of retroperitoneal adipose tissue; SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats; S, fat sum * Values are expressed as mean 6 SD (n ¼ 7 per group). y Lee index ¼ body weight1/3(g)/nasal–anal length (cm) 3 1000. z P < 0.01 compared with SN and TN groups. x P < 0.05 compared with SN and SCa groups.
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Table 3 Intestinal morphometry: villi height, crypt depth, muscular coat thickness, total wall, and number of goblet cells in SN, SCa, TN, and TCa rats* SN Villi height (mm) Crypt depth (mm) Muscular-coat thickness (mm) Total wall (mm) Goblet cells (50 images/animal)
SCa
379.53 6 45.90 164.83 6 21.67 76.67 6 19.87 616.70 6 55.77 233.80 6 40.41
y
530.80 6 50.70 205.02 6 20.65y 87.70 6 16.18 726.02 6 53.20y 194.50 6 36.30y
TN
TCa
407.91 6 68.35 195.10 6 19.24 104.07 6 16.85z 616.74 6 83.80 216.00 6 35.83
540.45 6 63.99y 204.60 6 26.05y 102.66 6 15.29z 721.18 6 72.38y 165.24 6 26.67y
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 5 per group). y P < 0.05 compared with SN and TN groups. z P < 0.05 compared with SN and SCa groups.
The histochemical periodic acid–Schiff reaction showed a significantly lower goblet cell population in the cafeteria groups (SCa and TCa) compared with the controls (SN and TN). Enzymatic analysis The activities of the enzymes alkaline phosphatase, lipase, b-galactosidase, maltase, and sucrase are presented in Table 4. A significant increase was observed in the enzymatic activities of alkaline phosphatase, lipase, and sucrase in animals that received the cafeteria diet. b-Galactosidase and maltase did not significantly differ among the groups. Quantitative analysis and morphometry of myenteric neurons The total myenteric population, quantified by the Giemsa method, did not vary between the groups, in contrast to the average number of myenteric neurons and mean neuronal area (square micrometers) of cell bodies (Table 5), indicating the lack of any effect due to obesity or physical training. Figure 1 shows the neuronal distribution according to size in the class intervals of 100 mm2 and indicates that the class interval between 101 and 200 mm2 was prevalent in the normally fed groups and that between 201 and 300 mm2 was for the cafeteria diet–fed groups. Discussion The cafeteria diet [4–8] administered to rats in the present study promoted the development of obesity and was charac-
terized as hyperlipidic [4–8], hypoproteic, and isocaloric. Reports describing high-lipid diets offered to rodents are found in the literature [4–7,13,28,29], including those investigating the maintenance of caloric consumption [7,28]. This model resembles human obesity, where diets with a high lipid value and an overload of carbohydrates and/or fat are the causative factors [4–8]. No differences were observed with reference to diet consumption and/or training, thus maintaining the normophagic behavior in all groups. The cafeteria diet in the present study did not stimulate higher food intake despite being described as highly palatable, thereby leading to hyperphagia [30] or hypophagia [8] based on caloric surplus. Physical training was not effective in changing the behavior of the animals, similar to the results obtained in mice trained on treadmills [31]; however, this result is in contrast to other studies in which rats ran on wheels [32] or swam despite consuming a hyperlipidic diet [8]. Thus, an anorexigenic effect attributed to physical activity [33] was not verified in the conditions under which the animals of this study were trained. The lack of intake reduction in terms of grams of the trained animals may be attributable to the long training period or intensity [33] to which they were subjected (90 d with moderate intensity), thereby facilitating adaptation and maintenance of a balanced consumption. In experiments in which physical training led to lower chow consumption, the training duration was short [32] or moderate [8]. The maintenance of rats on the cafeteria diet in the present study for a period of 120 d provided excess lipid [4] and reduced fiber [6], thus promoting an increase in body weight [4–7,28]. The normally fed animals had a lower percentage of lipid intake and higher percentage of fiber intake compared
Table 4 Enzymatic activity in SN, SCa, TN, and TCa rats* SN Alkaline phosphatase (U/g jejunum) Lipase (U/g jejunum) Sucrase (U/g jejunum) b-galactosidase (U/g jejunum) Maltase (U/g jejunum)
5.34 6 0.68 6.79 6 1.37 51.94 6 5.24 16.71 6 4.84 308.10 6 59.52
SCa y
10.30 6 2.06 7.94 6 0.97y 69.48 6 6.18y 12.35 6 1.96 344.60 6 52.82
TN
TCa
5.22 6 1.08 5.16 6 0.42 49.50 6 5.03 14.17 6 2.51 291.20 6 78.36
9.62 6 2.72y 8.55 6 2.13y 69.65 6 7.17y 16.27 6 4.00 299.60 6 74.44
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 5 per group). y P < 0.01 compared with SN and TN groups.
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Table 5 Myenteric neurons in an area of 17.92 mm2 and neuronal cell body area in SN, SCa, TN, and TCa rats*
2
Myenteric neurons in 17.92-mm area Neuronal cell body area (mm2)
SN
SCa
TN
TCa
7276.2 6 1338.4 199.9 6 63.6
6938.1 6 917.2 240.1 6 68.0
6709.5 6 1422.5 204.5 6 63.1
6829.1 6 1053.1 217.3 6 60.4
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 7 per group).
with those on the cafeteria diet. Fiber leads to satiety because of the distention mechanism of the stomach, thus increasing intestinal motility and elimination of nutrients, in addition to contributing to decreased body weight. The final body weights corresponded with measurements of the nasal–anal lengths, which were used to calculate the Lee indexes [21] (analogous to BMI). The Lee index was significantly high, proving the obesity of the investigated animals. Similar results have been found previously in obese rodents [12,13]. No influence of physical training was found with reference to body weight in the normally fed groups [34] or in those that received a hyperlipidic diet combined with physical training [29]. Weight of the adipose tissue is also an indicator of obesity. The cafeteria diet leads to an increase in periepididymal and retroperitoneal adipose tissue weights and in the sum of these tissue weights, consistent with previous reports [4–7]. This increase is directly related to the tissue cellularity. A diet rich in carbohydrates and/or lipids promotes increases in cell size and the number of adipocytes [2]. With reference to training of the TN or TCa groups, the deposits of adipose tissue were reduced before the end of the experiment. The data related to the TCa group showed an interaction between physical training and diet. This interaction was significantly different for the adipose tissue mass, where the physical exercise was able to attenuate the deleterious effect of the diet. A statistically significant reduction was also found with a simultaneous combination of the hyperlipidic diet and treadmill running [29] or swimming [7] in rats, thus indicating a positive effect of physical training and the
consequent importance of physical exercise in the control of adipose tissue mass. Intestinal morphometric parameters The cafeteria diet promoted an increase in the length of the small intestine. The values obtained in this experiment are similar to those described in the literature for rats that became obese after intake of the cafeteria diet introduced after the postweaning period until 90 d of age; those rats showed a significant increase in small intestine length, which is justified because of the typical reduction in the fiber content, increase in storage capacity, and caloric intake [6]. A lack of effect of physical training on the intestinal length was also observed, consistent with results obtained for rats trained in treadmill running for 180 d [35], suggesting that the same rats would not show any alteration after a prolonged training period. When the complete jejunal wall was evaluated, a significant increase was found in its thickness in the cafeteria group, without any apparent influence of the physical training. This difference may be a consequence of the tissue response of the mucosa or muscular coat, which are very sensitive to environmental influences in the intestinal lumen, mainly the intestinal mucosa [10,36]. When nutrients are provided, the intestinal mucosa is maintained; the mucosa tends to atrophy when nutrients are absent, with kinetic reductions in the intestinal crypts [10] and reductions in villi height and crypt depth in rats fed a hypoproteic diet [36]. The response involving villi height and crypt depth significantly increased in animals that received the cafeteria diet
Frequencies of the cellular body areas
450 400 350 300 250 200 150 100 50 0 0-100
101-200
201-300
301-400
401-500
501-600
Cellular body areas (µm2) Fig. 1. Frequency distribution of cellular body areas of myenteric neurons (700 neurons/group), classified in intervals of 100 mm2, in the following groups of rats: sedentary normally fed rats (white bars), sedentary rats fed a cafeteria diet (black bars), trained normally fed rats (speckled bars), and trained animals fed a cafeteria diet (striped bars).
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compared with the control animals. These results suggest that the type of aliment, and not the consumed amount, was responsible for the hypertrophic effect observed in the villi and crypts in the jejunum of obese animals. This hypothesis has been corroborated by other studies [37] that verified the increase in villi height in the jejunum and mucosal protein content in response to a high-lipid (70%) diet that led to increases in digestion and surface of absorption. Notably, compared with the control, the cafeteria diet in this study showed a difference of 75.34% in the lipid percentage. Lipid concentrations stimulated increases in villi height and crypt depth in SCa and TCa animals. The reduced protein concentration could lead to a decrease in the height and surface area of jejunum villi in rats [38]. Although the protein content in the diet used for this study was low, the influence from the lipid surplus may be a determinant for the response observed. The increase in the intestinal surface area of cafeteria animals (SCa and TCa) caused by the presence of increased villi and crypts suggests that the capacity of absorption was maximized, justifying the weight gain of these animals and the fact that physical training in this study did not reverse this condition. The increase in muscular-coat thickness in the jejunum in response to physical training was observed independently of the nutritional condition, with a possible influence on intestinal motility [39]. The production of free radicals, under conditions of short [40] and long [41] training with different intensities, in the smooth musculature of the organs are discussed in depth in the literature and are suggested to be responsible for these alterations. Smaller numbers of goblet cell populations were estimated in the jejunum of animals from the cafeteria groups (SCa and TCa). Goblet cells are involved in the production of mucous that protects and lubricates the intestinal epithelium surface, histochemically proved by periodic acid–Schiff staining. Although villi height and crypt depth were increased in obese animals, this condition was not reflected in the overall goblet cell population, indicating the involvement of other factors with the kinetics of this cell type. One of these factors may be the consistency of food in the cafeteria diet, characterized as being smooth, with a higher fat content and lower fiber content, compared with the pellets offered to the normally fed control groups. Different textures and higher fiber content would demand greater mucous secretion [42]. The protein level of the diet may be another factor to be considered. The increase in the number of goblet cells due to a high-protein diet has been previously reported in fish [43]. A reduction in their number due to a hypoproteic diet has been found in rats [44], and maintenance of goblet cell number independent of protein content in the diet has been found in mice [45]. The cafeteria diet used in the present study was characterized as hypoproteic with a low-fiber content, leading to lower mucous production. Two factors may have contributed to the reduction in goblet cell population observed in the SCa and TCa groups.
Enzymatic intestinal parameters The measurement of intestinal enzyme activity is an important tool for understanding the physiology of the intestine under normal and pathologic conditions [46]. Enzymatic activity can vary according to the level of substrates in the diet [13] and nutrient conditions, such as starvation or availability of food [47]. The activities of alkaline phosphatase and lipase are modulated by the presence of dietary lipids [13]. Increased alkaline phosphatase activity was observed in obese rats after consumption of a hyperlipidic diet [13] and after MSG administration [47]. This increase is justified because alkaline phosphatase is involved in the modulation of fat tissue in a few types of obesity, in which fat and body weight cannot be explained simply by hyperphagia [13,47]. Notably, in the present experiments, cafeteria diet–fed rats (hyperlipidic) showed normophagic behavior. No influence of diet or training was found on the lactase and maltase activities. High-carbohydrate diets, independent of whether the administration regimen was short (7 d in rats) [48] or prolonged (385 d in mice) [49], increased the activity of sucrase, corroborating the results of this study. The difference in the response of intestinal sucrase, with reference to the other two enzymes, maltase and lactase, could be attributed to the higher amount of the respective substrate in the diet. This evaluation was, however, not carried out in the present study, although considering the aliment type provided, sucrose was the most predominant substrate, followed by maltose—which are present in high, but insignificant, levels in the cafeteria diet. Intestinal intrinsic innervation The intrinsic innervation of the jejunum was evaluated through quantification and morphometry of the total myenteric population using whole-mount preparations stained with the Giemsa method [12,14,26,27] based on the affinity of methylene blue for the acidic compounds that are found in the neuronal cytoplasm (rough endoplasmic reticulum and free ribosome). Although quantification in the jejunum had been carried out in the intermediate and antimesenteric region to guarantee homogeneity of the results [27], there was no statistical difference between them. For this reason, the results were pooled. Variations were observed in the total myenteric populations with quantitative reductions in models involving aging [14,15], diabetes [17], and caloric restriction [18]; furthermore, increased neuron number was noted in experimental models of denervation [16] and obesity induced by MSG [12]. The maintenance of myenteric neuron number observed in the present obesity models, although apparently divergent, cannot be considered as being discrepant with other studies [12] that reported a larger neuron number in the ileum of rats that became obese due to MSG administration. These previous studies attributed the difference to the lower
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physical growth of obese animals that possessed smaller intestines and reduced muscular-coat thickness, thus resulting in a higher concentration of neurons [12]. The effect of a cafeteria diet on the intestines of obese rats was different from that observed in the MSG model, because the small intestine was longer, leading to greater neuronal dispersion. This observation supports the results obtained in the present experiment. The present results indicate the maintenance of the myenteric population because of the consumption of the cafeteria diet, but other models of caloric restriction [18] that lead to stress reduction have yielded positive results in terms of myenteric neuronal preservation. The absence of effects in the cafeteria diet group was also observed in relation to the neuronal body area. A similar response to the same technique was obtained for the ileum of rats in the experimental obesity model induced by neonatal administration of MSG [12]. Among the factors involved in the variation of the cell body area is oxidative stress, observed in the stomach [50] and ileum [17] of diabetic rats, a condition that was proven to be related to the formation of reactive oxygen species. Some investigators [5,51] have certified that diets with high fat and lower fiber levels promote the generation of free radicals; however, the results obtained in this study indicate that, if this condition is reproduced in the regular diet, then it would not affect the area of the neuronal body of myenteric neurons in the jejunum. In trained obese rats, a tendency toward reduction in the cellular area was observed when compared with obese animals. The maintenance of physical training for 8 wk by rats fed on a cafeteria diet resulted in increased plasma levels of antioxidants [8]. Reductions in the neuronal area in response to 180 d of physical training were observed [19] in aged normally fed rats compared with rats that did not undergo physical training. Regular physical activity can promote higher neuronal resistance to free radicals that are produced during the aging process. The extended time of physical training in normally fed animals could be the reason for this modified response. Conclusion In summary, the experimental cafeteria diet model is efficient at inducing obesity in rodents, with significant consequences on the morphometric and enzymatic intestinal parameters. Obesity caused increases in the length of the small intestine, villi height, crypt depth, whole-wall thickness, and enzymatic activities of alkaline phosphatase, lipase, and sucrase, in addition to a reduction in the number of goblet cells. Acknowledgments The authors thank Ana Paula de Santi Rampazzo, Maria dos Anjos Fortunato, and Maria Euride Cancino for their technical support.
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Basic nutritional investigation
www.nutritionjrnl.com
Effects of cafeteria diet on the jejunum in sedentary and physically trained rats Ce´lia Regina Scoaris, M.D.a,*, Gabriela Vasconcelos Rizo, M.D.a, Luciana Patrı´cia Roldi, M.D.a, Solange Marta Franzo´i de Moraes, Ph.D.a, Andre´ Ricardo Gomes de Proenc¸a, M.Sc.a, Rosane Marina Peralta, Ph.D.b, and Maria Raquel Marc¸al Natali, Ph.D.a a
Department of Morphophysiological Sciences, State University of Maringa´, Maringa´, Parana´, Brazil b Department of Biochemistry, State University of Maringa´, Maringa´, Parana´, Brazil Manuscript received December 4, 2008; accepted April 15, 2009.
Abstract
Objective: The effects of a cafeteria diet on the small intestine were investigated in adult Wistar rats under sedentary conditions and after physical training. Methods: Parameters including morphometry, enzyme activities, and total myenteric populations in the jejunum were evaluated. Results: The cafeteria diet, characterized as hyperlipidic, produced obese rats, corroborated by increases in the Lee index and the weights of the periepididymal and retroperitoneal adipose tissues (P < 0.01). Obesity caused increases in the length of the small intestine, villi height, crypt depth, whole-wall thickness (P < 0.05), and the enzymatic activities of alkaline phosphatase, lipase, and sucrase (P < 0.01), in addition to a reduction in the number of goblet cells (P < 0.05). With reference to the jejunal intrinsic innervations, the total number and area of myenteric neurons was unchanged regardless of the group. Physical training promoted 1) a reduction of the weight in the retroperitoneal and periepididymal adipose tissues (P < 0.05) and 2) an increase in the thickness of the muscular layer (P < 0.05). Conclusion: The cafeteria diet promoted obesity in rodents, leading to alterations in morphometry and enzymatic intestinal parameters, which were partily attenuated by physical training. Ó 2010 Elsevier Inc. All rights reserved.
Keywords:
Cafeteria diet; Intestinal morphometry; Jejunum; Myenteric neurons; Obesity; Physical training
Introduction The increasing incidence of obesity is a serious public health issue worldwide. According to the World Health Organization, there are more than 1 billion overweight adults with a body mass index (BMI) 25 kg/m2 and around 300 million people with a BMI 30 kg/m2 [1]. The etiology of obesity is extremely complex because of the involvement of neurologic, endocrine, genetic, and behavioral factors. Obesity is characterized by increased energy storage and This work was financed by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico and Fundac¸a˜o Arauca´ria. *Corresponding author. Tel.: þ55-44-3261-4704; fax: þ55-44-32228866. E-mail address: [email protected] (C. R. Scoaris). 0899-9007/10/$ – see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.04.012
hypertrophic and hyperplastic alterations in adipocytes, leading to weight gain from an energy imbalance [2]. Health complications attributable to obesity frequently described in the literature include diabetes, cardiovascular disease, orthopedic and postural alterations, breathing and emotional disorders, hepatic steatosis, and gastrointestinal alterations, which affect the quality of life and increase the risk of death. The current obesity epidemic requires a change in life habits, considered from an evolutionary point of view. Previously, men were adapted to periods of physical effort for survival because of a lack of food and the need to obtain fresh food and fiber. At present, humanity leads a more sedentary life, associated with industrialized, refined aliments, saturated fat, and low fiber. The impact of obesity can be decreased through regular physical activity, restoration of
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a positive energy balance, promotion of antioxidant defense mechanisms, and by decreasing the occurrence of diseases associated with oxidative stress [3]. Experimental animal models resembling human obesity include those in which a hyperlipidic diet (also referred to as a cafeteria diet), Western diet, or fast-food diet is used [4–8]. This diet, although with increased energy value, has low nutritive value, because it is overloaded with carbohydrates, fat, or both [4]. This type of diet is characterized by its capacity to induce not only increased lipid storage in the adipose tissue but also increased oxidative stress throughout the system [5]. The small intestine is responsible for digesting and absorbing nutrients and is structurally adapted to carry out these functions. It has a large surface area and functions in triggering signals to the central nervous system and ensuring energy homeostasis [9]. The intestinal mucosa is sensitive to alterations in this environment, with hypertrophy when there is food overload and atrophy when there is a lack of food [10]. In obesity conditions induced by the administration of monosodium glutamate (MSG), intestinal morphometric parameters, such as the villi, crypts, and muscular-coat thickness, were maintained in the jejunum of mice [11] and ileum of rats [12]. Enzymatic activity can be altered according to the alimentary substrate. Increased activity of alkaline phosphatase was found in the jejunum of obese rats due to the intake of a hyperlipidic diet [13]. The small intestine is extrinsically innervated by the autonomic nervous system, and its activity is modulated intrinsically by the enteric nervous system. This system is organized into a complex network of nerve fibers and neuronal cell bodies (e.g., interneurons, motor and sensory neurons). The myenteric plexus is one of the main ganglionated plexuses comprising the enteric nervous system, which is located between the inner circular muscular coat and the outer longitudinal muscular coat of the digestive tract and responds to intestinal motility, among other functions [9,12,14–19]. Morphologic and quantitative variations in the enteric neurons occur according to age [14,15], denervation model [16], diabetes [17], diet restriction [18], obesity induced by neonatal administration of MSG [12], and physical training [19]. Because myenteric neurons are sensitive to alterations in the tension of the intestinal wall and the chemical environment of the intestine, the present study aimed to evaluate morphometric and enzymatic parameters, in addition to the population of myenteric neurons, in the jejunum of obese rats, which were sedentary or subjected to physical training. Materials and methods Animals The present study used male 70-d-old Wistar rats (Rattus norvegicus) with an initial average weight of 270 g from the Animal House of the State University of Maringa´. All animal
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procedures of the study were approved by the committee of ethics on animal experimentation of the State University of Maringa´. According to the diet type and physical activity, 28 animals were allocated to four groups: sedentary normally fed rats (SN), sedentary rats fed a cafeteria diet (SCa), trained normally fed rats (TN), and trained rats fed a cafeteria diet (TCa). During the experimental period of 120 d, animals were housed in polypropylene boxes at room temperature (24 6 2 C) under a 12-h light/12-h dark cycle. Diet Animals from the SN and TN groups received standard rodent chow (Nuvilab-Nuvital (Curitiba, PR, Brazil), recommended by the National Research Council and National Institutes of Health, Bethesda, MD, USA) and water ad libitum. Animals from the cafeteria groups (SCa and TCa) received a cafeteria diet [4–8] consisting of the usual pellets of standard diet, cheese or bacon-flavored chips, bread, chocolate, marshmallows, peanut candy, filled cookies, wafer cookies, sausage, and mortadella, in addition to ad libitum soda and water. The standard rodent diet and cafeteria diet were analyzed in terms of the percentage of macronutrients and fiber content by the Laboratory of Animal Nutrition, Zootechny Department, Maringa´ State University. Diet consumption, in grams and calories, was measured daily, and liquid consumption and body weight were measured three and two times per week, respectively. Physical training After 30 d of feeding on a cafeteria diet, animals were subjected to physical training for a period of 12 wk, consisting of treadmill running (Inbrasport, Porto Alegre, Brazil) from 16:30 to 18:30 h, with a previous training adaptation period of 10 min, at speeds ranging from 0.3 to 0.6 km/h. The training protocol [20] consisted of running five times per week, ranging from 15 to 60 min of training per session, with a variable speed from 0.3 to 1.4 km/h and no change of treadmill tilt. Tissue collection When animals were 190 d old, they were weighed, anesthetized with sodium thiopental (Thionembutal (Sa˜o Paulo, Brazil), 40 mg/kg, intraperitoneally), and their nasal–anal length was measured to obtain the Lee index (body weight1/3 [g]/nasal–anal length [cm] 3 1000) [21]. Subsequently, laparotomy was carried out and the periepididymal and retroperitoneal adipose tissues were dissected out from the small intestine; the weights and lengths (from the pylori to the ileocecal junction) of these dissected samples were measured. The jejunum was isolated, its width was measured, and it was divided into samples, which were further subjected to the following protocols: histologic processing and
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embedding in paraffin for the morphometric study of the muscular coat and total wall; inclusion in historesin for the morphometric evaluation of intestinal crypts and villi; histochemistry to evaluate goblet cell population; analysis of intestinal enzymes (alkaline phosphatase, lipase, b-galactosidase, maltase, and sucrase); and whole-mount preparations to evaluate the total myenteric population. Morphometric analysis of the jejunal wall Jejunal samples of five animals from each group were collected, opened along the mesenteric insertion, washed with saline solution, and adhered in polystyrene. Samples were fixed in Bouin’s solution for 6 h, dehydrated using gradedconcentration solutions of ethanol (70%, 80%, 90%, and 100%), diaphanized in xylol, and embedded in histologic paraffin. Transverse semi-serial sections of 5-mm thickness were obtained using a Leica RM 2145 microtome (Leica Microsystems, Wetzlar, Germany) with a steel knife. The histologic sections were stained with hematoxylin and eosin to evaluate the muscular coat and intestinal wall (from the top of the villi to the serosa coat). Subsequently, 50 points each (micrometers) were measured at random in the muscular coat and intestinal wall per animal. Morphometric analysis of intestinal villi, crypts, and goblet cell populations To evaluate villi height, crypt depth, and total population of goblet cells, the jejunum samples of five animals per group were collected and opened along the mesenteric insertion and fixed in Bouin’s solution. After fixation, they were dehydrated and embedded in 2-hydroxy-methacrylate resin (Leica Microsystems). Transverse semi-serial sections of 2-mm thickness were obtained using a Leica RM 2145 microtome; the sections were stained with hematoxylin and eosin to measure the height of 90 villi and the depth of 90 crypts per animal. Semi-serial sections were also subjected to the histochemical periodic acid–Schiff staining technique to quantify the goblet cell population in 50 microscopic fields (0.352 mm2) per animal from 10 histologic sections. Morphometric analysis was carried out using the images obtained from a highresolution camera (Q Color 3 Olympus American, Burnaby, BC, Canada) coupled to an Olympus BX 41 microscope (Olympus, Tokyo, Japan); subsequently, these images were transmitted to a computer using Q Capture Pro 5.1 and Image-Pro Plus 4.5 (Media Cybernetics, Silver Springs, MD, USA). Intestinal enzyme levels Jejunal samples of five animals from each group were collected, frozen in liquid nitrogen, and stored in a freezer at 80 C. Samples were weighed, cut out, macerated with treated sand, suspended in 4 mL of sodium phosphate buffer
(50 mM, pH 6.5), and centrifuged for 15 min at 4 C at 4000 rpm. The supernatant was further used to determine the levels of alkaline phosphatase [22], lipase [23], sucrase [24], b-galactosidase [22], and maltase [25]. For all enzymes, a unit of enzymatic activity was defined as the enzyme quantity that produced 1.0 mmol of product per milliliter within 1 h under experimental conditions. The specific activity was expressed as units per gram of jejunum (wet weight). Whole-mount preparations Jejunal samples of seven rats from each experimental group subjected to whole-mount preparations were used for this study. The samples were stained by the Giemsa method [26] that allows estimations of the total myenteric population. Samples were excised, and the lumen, after washing with saline solution, was filled with Giemsa fixer using a syringe. The edges were tied to form a ‘‘balloon’’ and immersed in the same solution for a period of 24 h. Subsequently, the samples were opened along their mesenteric border and microdissected under a transillumination stereomicroscope, removing the mucosa and submucosa coats, while simultaneously preserving the muscular and serosa coats, to obtain the whole-mount preparations. These samples were stained with Giemsa stain, consisting mainly of methylene blue in 0.1 N Sorensen buffer (pH 6.9), under agitation for 24 h. The samples were then dehydrated in alcohol, diaphanized in xylol, assembled over slides, and coverslipped with Permount synthetic resin. Quantitative analysis and neuronal morphometry The myenteric neurons present in 80 microscopic fields were randomly quantified using an optical microscope (Olympus) with a 403 objective for the whole-mount preparations in the intermediate (60–120 and 240–300 degrees) and antimesenteric (120–240 degrees) regions, with reference to the mesenteric insertion of the intestinal circumference [27]. Each field corresponded to 0.224 mm2, totaling 17.92 mm2/animal. The measurements (square micrometers) of the cell bodies of 100 neurons/animal, for a total of 700 neurons per studied group, were obtained using computerized image analysis (Image Pro Plus 4.5, Media Cybernetics). The mean value 6 standard deviation for each group was obtained, and the neurons were distributed in class intervals of 100 mm2 according to neuronal area. Statistical analysis Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA). Two-way analysis of variance was followed by Bonferroni’s post hoc test to compare the mean values. Levels of p < 0.05 were considered statistically significant. Results are presented as mean 6 standard deviation.
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Table 1 Alimentary consumption, caloric consumption, percentages of protein, lipid, and fiber; and consumption of daily water, soda, and total liquid in SN, SCa, TN, and TCa rats*
Consumption (g) Consumption (kcal) Protein (%)y Lipids (%)y Fiber (%)y Water (mL) Soda (mL) Total liquid (mL)
SN
SCa
TN
TCa
25.92 6 2.52 101.10 6 8.83 22.23 4.00 5.73 40.19 6 10.55 — 40.19 6 10.55
25.32 6 4.41 89.63 6 14.63 9.98 16.22 1.46 14.98 6 7.09 24.62 6 13.40 39.60 6 16.69
24.41 6 1.79 95.24 6 7.01 22.23 4.00 5.73 39.90 6 9.87 — 39.9 6 9.87
26.65 6 5.02 95.48 6 15.88 9.98 16.22 1.46 13.72 6 6.57 27.22 6 13.11 40.94 6 14.65
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 7 per group). y Laboratory of Animal Nutrition, Zootechny Department, Maringa´ State University.
and thereby produced obese rats. Physical training did not attenuate this condition. The highest weight in the adipose tissues (periepididymal and retroperitoneal), in addition to their sum, was found in the SCa and TCa groups compared with the other groups. Obesity was confirmed in these rodents. The weights of the periepididymal and retroperitoneal adipose tissues, and their sum, were reduced in trained animals regardless of their nutritional condition.
Results Alimentary consumption and macronutrient percentage The results refer to alimentary, caloric, and liquid consumption, in addition to percentages of the macronutrients and fiber content of the diets (Table 1). The cafeteria diet is characterized as hyperlipidic, hypoproteic, and isocaloric, associated with reduced fiber content. The lack of significant differences in the alimentary intake in all groups indicates that diet and physical training did not alter the alimentary intake of these animals and that they were, hence, normophagic. Total liquid consumption was similar among all the groups studied; however, consumption of soda by the SCa and TCa groups was higher compared with that of water.
Intestinal morphometry The values obtained for intestinal length in the SN, SCa, TN, and TCa groups, respectively, were 118.86 6 6.04, 124.43 6 5.26, 121.57 6 3.46, and 124.43 6 3.26 cm. The values obtained for intestinal width were 1.01 6 0.38, 0.87 6 0.4, 0.80 6 0.18, and 0.86 6 0.15 cm, respectively. The lengths of the small intestine were significantly different in the groups that received the cafeteria diet. The morphometric results regarding villi height, crypt depth, intestinal total wall, and muscular-coat thickness and the quantification of total goblet cell population are presented in Table 3. The cafeteria diet increased the villi height, crypt depth, and total wall thickness. Physical training did not influence these parameters. Physical training promoted a significant increase in the muscular coat in animals of the physical training groups compared with the control group.
Validation of the obesity model Results validating the induction of the obesity condition subsequent to consumption of a cafeteria diet are presented in Table 2. Animals that consumed this diet for 120 d gained more weight compared with normally fed animals. Physical training was not effective in reducing or controlling weight gain. Trained TN and TCa groups were not different in terms of weight from the SN and SCa groups. The Lee index, a parameter considered analogous to the BMI, was high in animals that received the cafeteria diet Table 2 FW, Lee index, PER and RET weights, and S in SN, SCa, TN, and TCa rats* SN FW (g) Lee indexy PER (g/100 g body weight) RET (g/100 g body weight) S (g)
422.30 6 39.50 304.60 6 7.40 1.29 6 0.40 1.48 6 0.34 2.77 6 0.70
SCa z
523.20 6 52.00 322.20 6 11.30z 2.30 6 0.52z 3.54 6 0.64z 5.84 6 0.82z
TN
TCa
393.90 6 37.70 303.90 6 5.40 0.89 6 0.13x 1.09 6 0.24x 1.98 6 0.34x
522.80 6 47.50z 320.10 6 7.00z 2.01 6 0.35zx 3.23 6 0.41zx 5.24 6 0.70zx
FW, final body weight; PER, weight of periepididymal adipose tissue; RET, weight of retroperitoneal adipose tissue; SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats; S, fat sum * Values are expressed as mean 6 SD (n ¼ 7 per group). y Lee index ¼ body weight1/3(g)/nasal–anal length (cm) 3 1000. z P < 0.01 compared with SN and TN groups. x P < 0.05 compared with SN and SCa groups.
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Table 3 Intestinal morphometry: villi height, crypt depth, muscular coat thickness, total wall, and number of goblet cells in SN, SCa, TN, and TCa rats* SN Villi height (mm) Crypt depth (mm) Muscular-coat thickness (mm) Total wall (mm) Goblet cells (50 images/animal)
SCa
379.53 6 45.90 164.83 6 21.67 76.67 6 19.87 616.70 6 55.77 233.80 6 40.41
y
530.80 6 50.70 205.02 6 20.65y 87.70 6 16.18 726.02 6 53.20y 194.50 6 36.30y
TN
TCa
407.91 6 68.35 195.10 6 19.24 104.07 6 16.85z 616.74 6 83.80 216.00 6 35.83
540.45 6 63.99y 204.60 6 26.05y 102.66 6 15.29z 721.18 6 72.38y 165.24 6 26.67y
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 5 per group). y P < 0.05 compared with SN and TN groups. z P < 0.05 compared with SN and SCa groups.
The histochemical periodic acid–Schiff reaction showed a significantly lower goblet cell population in the cafeteria groups (SCa and TCa) compared with the controls (SN and TN). Enzymatic analysis The activities of the enzymes alkaline phosphatase, lipase, b-galactosidase, maltase, and sucrase are presented in Table 4. A significant increase was observed in the enzymatic activities of alkaline phosphatase, lipase, and sucrase in animals that received the cafeteria diet. b-Galactosidase and maltase did not significantly differ among the groups. Quantitative analysis and morphometry of myenteric neurons The total myenteric population, quantified by the Giemsa method, did not vary between the groups, in contrast to the average number of myenteric neurons and mean neuronal area (square micrometers) of cell bodies (Table 5), indicating the lack of any effect due to obesity or physical training. Figure 1 shows the neuronal distribution according to size in the class intervals of 100 mm2 and indicates that the class interval between 101 and 200 mm2 was prevalent in the normally fed groups and that between 201 and 300 mm2 was for the cafeteria diet–fed groups. Discussion The cafeteria diet [4–8] administered to rats in the present study promoted the development of obesity and was charac-
terized as hyperlipidic [4–8], hypoproteic, and isocaloric. Reports describing high-lipid diets offered to rodents are found in the literature [4–7,13,28,29], including those investigating the maintenance of caloric consumption [7,28]. This model resembles human obesity, where diets with a high lipid value and an overload of carbohydrates and/or fat are the causative factors [4–8]. No differences were observed with reference to diet consumption and/or training, thus maintaining the normophagic behavior in all groups. The cafeteria diet in the present study did not stimulate higher food intake despite being described as highly palatable, thereby leading to hyperphagia [30] or hypophagia [8] based on caloric surplus. Physical training was not effective in changing the behavior of the animals, similar to the results obtained in mice trained on treadmills [31]; however, this result is in contrast to other studies in which rats ran on wheels [32] or swam despite consuming a hyperlipidic diet [8]. Thus, an anorexigenic effect attributed to physical activity [33] was not verified in the conditions under which the animals of this study were trained. The lack of intake reduction in terms of grams of the trained animals may be attributable to the long training period or intensity [33] to which they were subjected (90 d with moderate intensity), thereby facilitating adaptation and maintenance of a balanced consumption. In experiments in which physical training led to lower chow consumption, the training duration was short [32] or moderate [8]. The maintenance of rats on the cafeteria diet in the present study for a period of 120 d provided excess lipid [4] and reduced fiber [6], thus promoting an increase in body weight [4–7,28]. The normally fed animals had a lower percentage of lipid intake and higher percentage of fiber intake compared
Table 4 Enzymatic activity in SN, SCa, TN, and TCa rats* SN Alkaline phosphatase (U/g jejunum) Lipase (U/g jejunum) Sucrase (U/g jejunum) b-galactosidase (U/g jejunum) Maltase (U/g jejunum)
5.34 6 0.68 6.79 6 1.37 51.94 6 5.24 16.71 6 4.84 308.10 6 59.52
SCa y
10.30 6 2.06 7.94 6 0.97y 69.48 6 6.18y 12.35 6 1.96 344.60 6 52.82
TN
TCa
5.22 6 1.08 5.16 6 0.42 49.50 6 5.03 14.17 6 2.51 291.20 6 78.36
9.62 6 2.72y 8.55 6 2.13y 69.65 6 7.17y 16.27 6 4.00 299.60 6 74.44
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 5 per group). y P < 0.01 compared with SN and TN groups.
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Table 5 Myenteric neurons in an area of 17.92 mm2 and neuronal cell body area in SN, SCa, TN, and TCa rats*
2
Myenteric neurons in 17.92-mm area Neuronal cell body area (mm2)
SN
SCa
TN
TCa
7276.2 6 1338.4 199.9 6 63.6
6938.1 6 917.2 240.1 6 68.0
6709.5 6 1422.5 204.5 6 63.1
6829.1 6 1053.1 217.3 6 60.4
SCa, sedentary rats fed a cafeteria diet; SN, sedentary normally fed rats; TCa, trained rats fed a cafeteria diet; TN, trained normally fed rats * Values are expressed as mean 6 SD (n ¼ 7 per group).
with those on the cafeteria diet. Fiber leads to satiety because of the distention mechanism of the stomach, thus increasing intestinal motility and elimination of nutrients, in addition to contributing to decreased body weight. The final body weights corresponded with measurements of the nasal–anal lengths, which were used to calculate the Lee indexes [21] (analogous to BMI). The Lee index was significantly high, proving the obesity of the investigated animals. Similar results have been found previously in obese rodents [12,13]. No influence of physical training was found with reference to body weight in the normally fed groups [34] or in those that received a hyperlipidic diet combined with physical training [29]. Weight of the adipose tissue is also an indicator of obesity. The cafeteria diet leads to an increase in periepididymal and retroperitoneal adipose tissue weights and in the sum of these tissue weights, consistent with previous reports [4–7]. This increase is directly related to the tissue cellularity. A diet rich in carbohydrates and/or lipids promotes increases in cell size and the number of adipocytes [2]. With reference to training of the TN or TCa groups, the deposits of adipose tissue were reduced before the end of the experiment. The data related to the TCa group showed an interaction between physical training and diet. This interaction was significantly different for the adipose tissue mass, where the physical exercise was able to attenuate the deleterious effect of the diet. A statistically significant reduction was also found with a simultaneous combination of the hyperlipidic diet and treadmill running [29] or swimming [7] in rats, thus indicating a positive effect of physical training and the
consequent importance of physical exercise in the control of adipose tissue mass. Intestinal morphometric parameters The cafeteria diet promoted an increase in the length of the small intestine. The values obtained in this experiment are similar to those described in the literature for rats that became obese after intake of the cafeteria diet introduced after the postweaning period until 90 d of age; those rats showed a significant increase in small intestine length, which is justified because of the typical reduction in the fiber content, increase in storage capacity, and caloric intake [6]. A lack of effect of physical training on the intestinal length was also observed, consistent with results obtained for rats trained in treadmill running for 180 d [35], suggesting that the same rats would not show any alteration after a prolonged training period. When the complete jejunal wall was evaluated, a significant increase was found in its thickness in the cafeteria group, without any apparent influence of the physical training. This difference may be a consequence of the tissue response of the mucosa or muscular coat, which are very sensitive to environmental influences in the intestinal lumen, mainly the intestinal mucosa [10,36]. When nutrients are provided, the intestinal mucosa is maintained; the mucosa tends to atrophy when nutrients are absent, with kinetic reductions in the intestinal crypts [10] and reductions in villi height and crypt depth in rats fed a hypoproteic diet [36]. The response involving villi height and crypt depth significantly increased in animals that received the cafeteria diet
Frequencies of the cellular body areas
450 400 350 300 250 200 150 100 50 0 0-100
101-200
201-300
301-400
401-500
501-600
Cellular body areas (µm2) Fig. 1. Frequency distribution of cellular body areas of myenteric neurons (700 neurons/group), classified in intervals of 100 mm2, in the following groups of rats: sedentary normally fed rats (white bars), sedentary rats fed a cafeteria diet (black bars), trained normally fed rats (speckled bars), and trained animals fed a cafeteria diet (striped bars).
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compared with the control animals. These results suggest that the type of aliment, and not the consumed amount, was responsible for the hypertrophic effect observed in the villi and crypts in the jejunum of obese animals. This hypothesis has been corroborated by other studies [37] that verified the increase in villi height in the jejunum and mucosal protein content in response to a high-lipid (70%) diet that led to increases in digestion and surface of absorption. Notably, compared with the control, the cafeteria diet in this study showed a difference of 75.34% in the lipid percentage. Lipid concentrations stimulated increases in villi height and crypt depth in SCa and TCa animals. The reduced protein concentration could lead to a decrease in the height and surface area of jejunum villi in rats [38]. Although the protein content in the diet used for this study was low, the influence from the lipid surplus may be a determinant for the response observed. The increase in the intestinal surface area of cafeteria animals (SCa and TCa) caused by the presence of increased villi and crypts suggests that the capacity of absorption was maximized, justifying the weight gain of these animals and the fact that physical training in this study did not reverse this condition. The increase in muscular-coat thickness in the jejunum in response to physical training was observed independently of the nutritional condition, with a possible influence on intestinal motility [39]. The production of free radicals, under conditions of short [40] and long [41] training with different intensities, in the smooth musculature of the organs are discussed in depth in the literature and are suggested to be responsible for these alterations. Smaller numbers of goblet cell populations were estimated in the jejunum of animals from the cafeteria groups (SCa and TCa). Goblet cells are involved in the production of mucous that protects and lubricates the intestinal epithelium surface, histochemically proved by periodic acid–Schiff staining. Although villi height and crypt depth were increased in obese animals, this condition was not reflected in the overall goblet cell population, indicating the involvement of other factors with the kinetics of this cell type. One of these factors may be the consistency of food in the cafeteria diet, characterized as being smooth, with a higher fat content and lower fiber content, compared with the pellets offered to the normally fed control groups. Different textures and higher fiber content would demand greater mucous secretion [42]. The protein level of the diet may be another factor to be considered. The increase in the number of goblet cells due to a high-protein diet has been previously reported in fish [43]. A reduction in their number due to a hypoproteic diet has been found in rats [44], and maintenance of goblet cell number independent of protein content in the diet has been found in mice [45]. The cafeteria diet used in the present study was characterized as hypoproteic with a low-fiber content, leading to lower mucous production. Two factors may have contributed to the reduction in goblet cell population observed in the SCa and TCa groups.
Enzymatic intestinal parameters The measurement of intestinal enzyme activity is an important tool for understanding the physiology of the intestine under normal and pathologic conditions [46]. Enzymatic activity can vary according to the level of substrates in the diet [13] and nutrient conditions, such as starvation or availability of food [47]. The activities of alkaline phosphatase and lipase are modulated by the presence of dietary lipids [13]. Increased alkaline phosphatase activity was observed in obese rats after consumption of a hyperlipidic diet [13] and after MSG administration [47]. This increase is justified because alkaline phosphatase is involved in the modulation of fat tissue in a few types of obesity, in which fat and body weight cannot be explained simply by hyperphagia [13,47]. Notably, in the present experiments, cafeteria diet–fed rats (hyperlipidic) showed normophagic behavior. No influence of diet or training was found on the lactase and maltase activities. High-carbohydrate diets, independent of whether the administration regimen was short (7 d in rats) [48] or prolonged (385 d in mice) [49], increased the activity of sucrase, corroborating the results of this study. The difference in the response of intestinal sucrase, with reference to the other two enzymes, maltase and lactase, could be attributed to the higher amount of the respective substrate in the diet. This evaluation was, however, not carried out in the present study, although considering the aliment type provided, sucrose was the most predominant substrate, followed by maltose—which are present in high, but insignificant, levels in the cafeteria diet. Intestinal intrinsic innervation The intrinsic innervation of the jejunum was evaluated through quantification and morphometry of the total myenteric population using whole-mount preparations stained with the Giemsa method [12,14,26,27] based on the affinity of methylene blue for the acidic compounds that are found in the neuronal cytoplasm (rough endoplasmic reticulum and free ribosome). Although quantification in the jejunum had been carried out in the intermediate and antimesenteric region to guarantee homogeneity of the results [27], there was no statistical difference between them. For this reason, the results were pooled. Variations were observed in the total myenteric populations with quantitative reductions in models involving aging [14,15], diabetes [17], and caloric restriction [18]; furthermore, increased neuron number was noted in experimental models of denervation [16] and obesity induced by MSG [12]. The maintenance of myenteric neuron number observed in the present obesity models, although apparently divergent, cannot be considered as being discrepant with other studies [12] that reported a larger neuron number in the ileum of rats that became obese due to MSG administration. These previous studies attributed the difference to the lower
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physical growth of obese animals that possessed smaller intestines and reduced muscular-coat thickness, thus resulting in a higher concentration of neurons [12]. The effect of a cafeteria diet on the intestines of obese rats was different from that observed in the MSG model, because the small intestine was longer, leading to greater neuronal dispersion. This observation supports the results obtained in the present experiment. The present results indicate the maintenance of the myenteric population because of the consumption of the cafeteria diet, but other models of caloric restriction [18] that lead to stress reduction have yielded positive results in terms of myenteric neuronal preservation. The absence of effects in the cafeteria diet group was also observed in relation to the neuronal body area. A similar response to the same technique was obtained for the ileum of rats in the experimental obesity model induced by neonatal administration of MSG [12]. Among the factors involved in the variation of the cell body area is oxidative stress, observed in the stomach [50] and ileum [17] of diabetic rats, a condition that was proven to be related to the formation of reactive oxygen species. Some investigators [5,51] have certified that diets with high fat and lower fiber levels promote the generation of free radicals; however, the results obtained in this study indicate that, if this condition is reproduced in the regular diet, then it would not affect the area of the neuronal body of myenteric neurons in the jejunum. In trained obese rats, a tendency toward reduction in the cellular area was observed when compared with obese animals. The maintenance of physical training for 8 wk by rats fed on a cafeteria diet resulted in increased plasma levels of antioxidants [8]. Reductions in the neuronal area in response to 180 d of physical training were observed [19] in aged normally fed rats compared with rats that did not undergo physical training. Regular physical activity can promote higher neuronal resistance to free radicals that are produced during the aging process. The extended time of physical training in normally fed animals could be the reason for this modified response. Conclusion In summary, the experimental cafeteria diet model is efficient at inducing obesity in rodents, with significant consequences on the morphometric and enzymatic intestinal parameters. Obesity caused increases in the length of the small intestine, villi height, crypt depth, whole-wall thickness, and enzymatic activities of alkaline phosphatase, lipase, and sucrase, in addition to a reduction in the number of goblet cells. Acknowledgments The authors thank Ana Paula de Santi Rampazzo, Maria dos Anjos Fortunato, and Maria Euride Cancino for their technical support.
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