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Basic nutritional investigation
Effects of cafeteria diet on the jejunum in sedentaryand 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 ratsunder sedentary conditions and after physical training.
Methods:
Parameters including morphometry, enzyme activities, and total myenteric populations inthe jejunum were evaluated.
Results:
The cafeteria diet, characterized as hyperlipidic, produced obese rats, corroborated by in-creases 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 su-crase (
 P
<
0.01), in addition to a reduction in the number of goblet cells (
 P
<
0.05). With reference tothe jejunal intrinsic innervations, the total number and area of myenteric neurons was unchanged re-gardlessofthegroup.Physicaltrainingpromoted1)areductionoftheweightintheretroperitonealandperiepididymal 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 morphometryand enzymatic intestinal parameters, which were partily attenuated by physical training.
Ó
2010Elsevier Inc. All rights reserved.
 Keywords:
Cafeteria diet; Intestinal morphometry; Jejunum; Myenteric neurons; Obesity; Physical training
Introduction
The increasing incidence of obesity is a serious publichealth issue worldwide. According to the World HealthOrganization, there are more than 1 billion overweight adultswith a body mass index (BMI)
!
25 kg/m
2
and around 300million people with a BMI
!
30 kg/m
2
[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 andhypertrophic and hyperplastic alterations in adipocytes, lead-ing to weight gain from an energy imbalance[2].Healthcomplicationsattributabletoobesityfrequentlyde-scribed in the literature include diabetes, cardiovascular disease, orthopedic and postural alterations, breathing andemotional disorders, hepatic steatosis,andgastrointestinalal-terations, which affect the quality of life and increase the riskof death.The current obesity epidemic requires a change in lifehabits, considered from an evolutionary point of view. Previ-ously, men were adapted to periods of physical effort for sur-vival because of a lack of food and the need to obtain freshfood and fiber. At present, humanity leads a more sedentarylife, associated with industrialized, refined aliments, satu-rated fat, and low fiber. The impact of obesity can bedecreased through regular physical activity, restoration of 
This work was financed by the Conselho Nacional de DesenvolvimentoCientı´fico e Tecnolo´gico and Fundac¸a˜o Arauca´ria.*Corresponding author. Tel.:
þ
55-44-3261-4704; fax:
þ
55-44-3222-8866.
 E-mail address:
celiareginabio@hotmail.com(C. R. Scoaris).0899-9007/10/$ – see front matter 
Ó
2010 Elsevier Inc. All rights reserved.doi:10.1016/j.nut.2009.04.012Nutrition 26 (2010) 312–320www.nutritionjrnl.com
 
a positive energy balance, promotion of antioxidant defensemechanisms, and by decreasing the occurrence of diseasesassociated with oxidative stress[3].Experimental animal models resembling human obesityinclude those in which a hyperlipidic diet (also referred toas a cafeteria diet), Western diet, or fast-food diet is used[4–8]. This diet, although with increased energy value, haslow nutritive value, because it is overloaded with carbohy-drates, fat, or both[4]. This type of diet is characterized byits capacity to induce not only increased lipid storage in theadipose tissue but also increased oxidative stress throughout the system[5].The small intestine is responsible for digesting and ab-sorbing nutrients andisstructurally adaptedtocarry out thesefunctions. It has a large surface area and functions in trigger-ing signals to the central nervous system and ensuring energyhomeostasis[9]. The intestinal mucosa is sensitive to alter-ations in this environment, with hypertrophy when there isfood overload and atrophy when there is a lack of food[10].In obesity conditions induced by the administration of monosodium glutamate (MSG), intestinal morphometric pa-rameters, such as the villi, crypts, and muscular-coat thick-ness, were maintained in the jejunum of mice[11]andileum of rats[12]. Enzymatic activity can be altered accord-ing to the alimentary substrate. Increased activity of alkalinephosphatasewasfoundinthejejunumofobeseratsduetotheintake of a hyperlipidic diet [13].The small intestine is extrinsically innervated by the auto-nomic nervous system, and its activity is modulated intrinsi-cally by the enteric nervous system. This system is organizedinto a complex network of nerve fibers and neuronal cellbodies (e.g., interneurons, motor and sensory neurons). Themyenteric plexus is one of the main ganglionated plexusescomprising the enteric nervous system, which is located be-tween the inner circular muscular coat and the outer longitu-dinal muscular coat of the digestive tract and responds tointestinal motility, among other functions[9,12,14–19].Morphologic and quantitative variations in the entericneurons 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 inthe tension of the intestinal wall and the chemical environ-ment of the intestine, the present study aimed to evaluatemorphometric and enzymatic parameters, in addition to thepopulation of myenteric neurons, in the jejunum of obeserats, which were sedentary or subjected to physical training.
Materials and methods
 Animals
The present study used male 70-d-old Wistar rats (
 Rattusnorvegicus
) with an initial average weight of 270 g from theAnimal House of the State University of Maringa´. All animalprocedures 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 ani-mals were allocated to four groups: sedentary normally fedrats (SN), sedentary rats fed a cafeteria diet (SCa), trainednormally fed rats (TN), and trained rats fed a cafeteria diet (TCa). During the experimental period of 120 d, animalswere housed in polypropylene boxes at room temperature(24
6
2
C) under a 12-h light/12-h dark cycle.
 Diet 
AnimalsfromtheSNandTNgroupsreceivedstandardro-dent chow (Nuvilab-Nuvital (Curitiba, PR, Brazil), recom-mended by the National Research Council and NationalInstitutes of Health, Bethesda, MD, USA) and water ad libi-tum. Animals from the cafeteria groups (SCa and TCa) re-ceived a cafeteria diet [4–8]consisting of the usual pelletsof standard diet, cheese or bacon-flavored chips, bread, choc-olate, marshmallows, peanut candy, filled cookies, wafer cookies, sausage, and mortadella, in addition to ad libitumsoda and water. The standard rodent diet and cafeteria diet were analyzed in terms of the percentage of macronutrientsand fiber content by the Laboratory of Animal Nutrition,Zootechny Department, Maringa´ State University.Diet consumption, in grams and calories, was measureddaily, and liquid consumption and body weight were mea-sured three and two times per week, respectively.
 Physical training
After 30 dof feeding ona cafeteria diet,animals were sub- jected to physical training for a period of 12 wk, consisting of treadmill running (Inbrasport, Porto Alegre, Brazil) from16:30 to 18:30 h, with a previous training adaptation periodof 10 min, at speeds ranging from 0.3 to 0.6 km/h. The train-ing protocol[20]consisted of running five times per week,rangingfrom15to60 minoftraining persession, withavari-able speed from 0.3 to 1.4 km/h and no change of treadmilltilt.
Tissue collection
When animals were 190 d old, they were weighed, anes-thetized with sodium thiopental (Thionembutal (Sa˜o Paulo,Brazil), 40 mg/kg, intraperitoneally), and their nasal–anallength was measured to obtain the Lee index (bodyweight 
1/3
[g]/nasal–anal length [cm]
3
1000)[21]. Subse-quently, laparotomy was carried out and the periepididymaland retroperitoneal adipose tissues were dissected out fromthe small intestine; the weights and lengths (from the pylorito the ileocecal junction) of these dissected samples weremeasured.Thejejunumwasisolated,itswidthwasmeasured,anditwasdividedintosamples,whichwerefurthersubjectedto the following protocols: histologic processing and
C. R. Scoaris et al. / Nutrition 26 (2010) 312–320
313
 
embedding in paraffin for the morphometric study of themuscular coat and total wall; inclusion in historesin for themorphometric evaluation of intestinal crypts and villi;histochemistry to evaluate goblet cell population; analysisof intestinal enzymes (alkaline phosphatase, lipase,
b
-galac-tosidase, maltase, and sucrase); and whole-mount prepara-tions to evaluate the total myenteric population.
 Morphometric analysis of the jejunal wall 
Jejunal samples of five animals from each group were col-lected, opened along the mesenteric insertion, washed withsaline solution, and adhered in polystyrene. Samples werefixed in Bouin’s solution for 6 h, dehydrated using graded-concentration solutions of ethanol (70%, 80%, 90%, and100%), diaphanized in xylol, and embedded in histologicparaffin. Transverse semi-serial sections of 5-
m
m thicknesswere obtained using a Leica RM 2145 microtome (Leica Mi-crosystems, Wetzlar, Germany) with a steel knife. The histo-logic sections were stained with hematoxylin and eosin toevaluate the muscular coat and intestinal wall (from the topof 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 populationofgoblet cells, the jejunum samples of five animals per groupwere collected and opened along the mesentericinsertion andfixed in Bouin’s solution. After fixation, they were dehy-drated and embedded in 2-hydroxy-methacrylate resin (Leica Microsystems). Transverse semi-serial sections of 2-
m
mthickness were obtained using a Leica RM 2145 microtome;the sections were stained with hematoxylin and eosin to mea-sure the height of 90 villi and the depth of 90 crypts per animal.Semi-serial sections were also subjected to the histochem-ical periodic acid–Schiff staining technique to quantify thegoblet cell population in 50 microscopic fields (0.352 mm
2
)per animal from 10 histologic sections. Morphometric analy-sis was carried out using the images obtained from a high-resolution camera (Q Color 3 Olympus American, Burnaby,BC, Canada) coupled to an Olympus BX 41 microscope(Olympus, Tokyo, Japan); subsequently, these images weretransmitted to a computer using Q Capture Pro 5.1 and Im-age-Pro Plus 4.5 (Media Cybernetics, Silver Springs, MD,USA).
 Intestinal enzyme levels
Jejunal samples of five animals from each group were col-lected, frozen in liquid nitrogen, and stored in a freezer at 
À
80
C. Samples were weighed, cut out, macerated withtreated 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 determinethe levels of alkaline phosphatase[22], lipase[23], sucrase [24],
b
-galactosidase[22], and maltase[25]. For all enzymes, a unit of enzymatic activity was definedastheenzymequantity thatproduced1.0
m
molofproductper milliliter within 1 h under experimental conditions. The spe-cific activity was expressed as units per gram of jejunum(wet weight).
Whole-mount preparations
Jejunal samples of seven rats from each experimentalgroup subjected to whole-mount preparations were used for this study. The samples were stained by the Giemsa method[26]thatallowsestimationsofthetotalmyentericpopulation.Samples were excised, and the lumen, after washing with sa-line solution, was filled with Giemsa fixer using a syringe.The edges were tied to form a ‘‘balloon’’ and immersed inthe same solution for a period of 24 h.Subsequently, the samples were opened along their mes-enteric border and microdissected under a transilluminationstereomicroscope, removing the mucosa and submucosa coats, while simultaneously preserving the muscular and se-rosa coats, to obtain the whole-mount preparations. Thesesamples were stained with Giemsa stain, consisting mainlyof methylene blue in 0.1 N Sorensen buffer (pH 6.9), under agitation for 24 h. The samples were then dehydrated in alco-hol, diaphanized in xylol, assembled over slides, and cover-slipped with Permount synthetic resin.
Quantitative analysis and neuronal morphometry
The myenteric neurons present in 80 microscopic fieldswere randomly quantified using an optical microscope(Olympus) with a 40
3
objective for the whole-mount prepa-rations in the intermediate (60–120 and 240–300 degrees)and antimesenteric (120–240 degrees) regions, with refer-ence to the mesenteric insertion of the intestinal circumfer-ence[27]. Each field corresponded to 0.224 mm
2
, totaling17.92 mm
2
 /animal. The measurements (square micrometers)of the cell bodies of 100 neurons/animal, for a total of 700neurons per studied group, were obtained using computer-izedimageanalysis(ImageProPlus4.5,MediaCybernetics).The mean value
6
standard deviation for each group was ob-tained, and the neurons were distributed in class intervals of 100
m
m
2
according to neuronal area.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism3.0 (GraphPad Software, San Diego, CA, USA). Two-wayanalysis of variance was followed by Bonferroni’s post hoctest to compare the mean values. Levels of 
p
<
0.05 wereconsidered statistically significant. Results are presented asmean
6
standard deviation.
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