Original Article

International Journal of Obesity (2015) 39, 430–437; doi:10.1038/ijo.2014.125; published online 12 August 2014

Animal Models

Cafeteria diet overfeeding in young male rats impairs the adaptive response to fed/fasted conditions and increases adiposity independent of body weight

H Castro1, C A Pomar1, C Picó1, J Sánchez1 and A Palou1

1Molecular Biology, Nutrition and Biotechnology (Nutrigenomics), University of the Balearic Islands (UIB), Cra, Palma de Mallorca and CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Spain

Correspondence: Professor A Palou, Universitat de les Illes Balears, Biología Molecular, Nutrición y Biotecnología (Nutrigenómica). Edifici Mateu Orfila, Carretera de Valldemossa Km 7.5, 07122 Palma de Mallorca, Spain. E-mail: andreu.palou@uib.es

Received 12 May 2014; Revised 8 July 2014; Accepted 13 July 2014
Accepted article preview online 21 July 2014; Advance online publication 12 August 2014

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Abstract

Objective:

 

We analyzed the effects of a short exposure to a cafeteria diet during early infancy in rats on their metabolic response to fed/fasting conditions in key tissues involved in energy homeostasis.

Methods:

 

Ten-day-old male pups were fed a control or a cafeteria diet for 12 days and then killed under ad libitum feeding conditions or 12h fasting. The expression of key genes related to energy metabolism in liver, retroperitoneal white adipose tissue (WAT) and hypothalamus were analyzed.

Results:

 

Despite no differences in body weight, cafeteria-fed animals had almost double the fat mass of control rats. They also showed higher food intake, higher leptinemia and altered hypothalamic expression of Neuropetide Y, suggesting a dysfunction in the control of food intake. Unlike controls, cafeteria-fed animals did not decrease WAT expression of Pparg, sterol regulatory element binding transcription factor 1 or Cidea under fasting conditions, and displayed lower Pnpla2 expression than controls. In liver, compared with controls, cafeteria animals presented: (i) lower expression of genes related with fatty acid uptake and lipogenesis under ad libitum-fed conditions; (ii) higher expression of fatty acid oxidation-related genes and glucokinase under fasting conditions; (iii) greater expression of leptin and insulin receptors; and higher protein levels of insulin receptor and the pAMPK/AMPK ratio.

Conclusion:

 

A short period of exposure to a cafeteria diet in early infancy in rat pups is enough to disturb the metabolic response to fed/fasting conditions in key tissues involved in energy homeostasis, particularly in WAT, and hence induces an exacerbated body fat accumulation and increased metabolic risk, with no apparent effects on body weight.

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Introduction

The prevalence of obesity and overweight in childhood has increased dramatically in recent decades, so have the risks of associated medical conditions, such as type 2 diabetes and cardiovascular disease, raising serious concerns for public health.1,2 The rise in childhood overweight and obesity is probably due to the changes in environmental and social factors. Obesity is encouraged by current obesogenic environmental conditions that promote the intake of high-energy-dense foods and increased portion sizes.3, 4, 5 Cafeteria and post-cafeteria animal models provide a useful tool to study the metabolic syndrome in humans. Exposure to a cafeteria diet in rats induces persistent hyperphagia and increased energy intake as a result of the variety and novelty of the foods available, similarly to unhealthy dietary patterns observed in human subjects.6, 7, 8 Previous results from our group and others show that cafeteria diet administered during the first months of life and adulthood leads to an important and persistent increase in body weight, associated with excessive body fat accumulation and metabolic alterations, which are not completely reverted when animals are moved to a control diet.8, 9, 10, 11

During early stages of development, rodents are more resistant to increase their weight and this has been explained by a greater capacity of adaptive brown adipose tissue-thermogenesis.12,13 However, an exacerbated accumulation of fat may occur in the absence of notorious body mass index or body weight increases, and this risk can be triggered by obesogenic feeding behavior, which may be particularly harmful at young ages.

In the obese state, profound metabolic disturbances exist that may change the ability to respond to different stressors. Of particular interest is the maintenance of nutrient homeostasis under the repeated sequences of fed/fasting conditions that characterize food habits in mammals, and the metabolic response to these situations involves hormonal and metabolic adaptations which are accompanied by changes in gene expression.14, 15, 16 Adult obese rats fed a cafeteria diet have been described to present impaired nutritional regulation, as the metabolic adaptations to acute changes in feeding conditions are impaired.17, 18, 19 However, although there is extensive literature on the long-term effects of cafeteria diet intake, little is known about the short-term effects of the intake of this obesogenic diet during early infancy and the molecular events initiated by this condition. Thus, the objective of this study was to evaluate the effects of a short period of cafeteria diet feeding during early infancy on the capacity to respond to acute changes in feeding conditions (fed/fasting states) in key metabolic tissues such as liver, white adipose tissue (WAT) and hypothalamus, and the impact this may have on energy homeostasis.

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Materials and methods

Ethical approval

The animal protocol followed in this study was reviewed and approved by the Bioethical Committee of our University (Ref 3513 (26/03/2012)) and guidelines for the use and care of laboratory animals of the University were followed.

Animals and experimental design

Virgin female Wistar rats (Charles River Laboratories Spain, SA, Barcelona, Spain), housed at 22°C with a period of light/dark of 12h (lights on from 0800 to 2000 hours) and with free access to food and water, were mated with male rats. At day 1 after delivery, excess pups in each litter were removed to keep 10 pups per dam. On day 10 of lactation, litters were randomly distributed into two dietary groups: control group, fed with a standard chow diet (3300kcalkg−1, Panlab, Barcelona, Spain) and cafeteria group, fed with a cafeteria diet in addition to the standard chow. The cafeteria feeding regimen used was based on that previously described,6,7 containing the following food items: cookies with liver pate and sobrasada (a typical Majorcan sausage), candies, fresh bacon, biscuits, chocolate, salted peanuts, cheese, milk containing 20% w/v) sucrose and ensaimada (a typical Majorcan pastry). Pups were weaned at day 21, and continued under control or cafeteria diet. Males from both control and cafeteria groups were killed at day 22 under ad libitum or fasting (overnight) conditions during the first hour of the light cycle. Food intake was recorded in ad libitum-fed animals during the last 12h before killing; in the case of cafeteria animals, the exact amount of each item eaten by animals was measured to know the exact composition of the food eaten. The hypothalamus, liver and various WAT depots—epididymal, retroperitoneal, mesenteric and inguinal—were rapidly removed, weighed and immediately frozen in liquid nitrogen and stored at −70°C. Blood was also collected and centrifuged at 1000g for 10min to collect the plasma, which was stored at −20°C until analysis. Body weight was recorded and body fat content (by EchoMRI-700, Echo Medical Systems, LLC., TX, USA) was measured on day 22.

Analyses of blood parameters

Blood glucose concentration was measured by Accu-Chek Glucometer (Roche Diagnostics, Barcelona, Spain). Plasma leptin concentration was measured using a mouse leptin ELISA (enzyme-linked immunosorbent assay) kit (R&D Systems, Minneapolis, MN, USA). Plasma insulin concentration was measured using an ultrasensitive rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden) following standard procedures. Commercial enzymatic colorimetric kits were used for the determination of plasma triglyceride levels (Sigma, Madrid, Spain) and non-esterified (or free) fatty acids (Wako Chemicals GmbH, Neuss, Germany).

RNA extraction

Total RNA was extracted from liver and retroperitoneal WAT (rpWAT) by E.Z.N.A. RNA Purification System (Omega Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer's instructions. From the hypothalamus, total RNA was extracted by Tripure Reagent (Roche Diagnostic Gmbh, Mannheim, Germany) according to the manufacturer’s instructions. Isolated RNA was quantified using the NanoDrop ND-1000 spectrophotometer (NadroDrop Technologies, Wilmington, DE, USA) and its integrity confirmed using agarose gel electrophoresis.

Real-time quantitative RT-PCR analysis

Real-time quantitative reverse transcriptase-PCR was used to measure mRNA expression levels of selected genes in the hypothalamus, liver and rpWAT. Specifically, insulin receptor (Insr), leptin receptor (Lepr), Ghrelin receptor, the suppressor of cytokine signaling 3 (Socs3), Signal transducer and activator of transcription 3 (Stat3), Neuropetide Y (Npy) and pro-opiomelanocortin (Pomc) in hypothalamus; Cd36, sterol regulatory element binding transcription factor 1 (Srebf1), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), peroxisome proliferator activated receptor alfa (Ppara), carnitine palmitoyl transferase 1a, liver (Cpt1a), pyruvate dehydrogenase kinase 4 (Pdk4), glucokinase (Gck), Lepr, Socs3, Insr and insulin receptor substrate 1 (Irs1) in liver; and Cd36, lipoprotein lipase, solute carrier family 2 (facilitated glucose transporter), member 4 (Slc2a4), hexokinase II (Hk2), Pparg, Srebf1, Fasn, Cell death activator CIDE-A (Cidea), patatin-like phospholipase domain containing 2 (Pnpla2), Cpt1b, leptin (Lep) and Insr in rpWAT. GDP dissociation inhibitor 1 (Gdi), in hypothalamus, and 18S, in liver and rpWAT, were used as reference genes.

Total RNA (0.25μg; in a final volume of 5μl) was denatured at 65°C for 10min and then reverse transcribed to cDNA using MuLV reverse transcriptase (Applied Biosystem, Madrid, Spain) at 20°C for 15min, 42°C for 30min, with a final step of 5min at 95°C in an Applied Biosystems 2720 Thermal Cycler (Applied Biosystem). Each PCR was performed from diluted cDNA template, forward and reverse primers (1μM each), and Power SYBER Green PCR Master Mix (Applied Biosystems). Primers were obtained from Sigma. Real-time PCR was performed using the Applied BiosystemsStepOnePlus Real-Time PCR Systems (Applied Biosystems) with the following profile: 10min at 95°C, followed by a total of 40 two-temperature cycles (15s at 95°C and 1min at 60°C). To verify the purity of the products, a melting curve was produced after each run according to the manufacturer’s instructions. The threshold cycle was calculated by the instrument's software (StepOne Software v2.2.2, Applied Biosystem) and the relative expression of each mRNA was calculated as previously described.20

Western blot analysis

Tissue was homogenized at 4°C in 1:5 (w:v) in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher, Rockford, IL, USA). Western blot was performed in a 4–15% Criterion TGX Precast Gel (BioRad, Madrid, Spain), and transferred to a nitrocellulose membrane. The primary antibodies used were the following: monoclonal rabbit anti-Insulin Receptor β (#3025), monoclonal rabbit anti-Akt (#9272), monoclonal mouse anti-AMPKα (#2793), monoclonal rabbit anti-pospho-AMPKα (#2535), all of these from Cell Signaling, Danvers, MA, USA. Membranes were also incubated with anti-beta-actin antibody to ensure the equal loading (#3700 from Cell Signaling). Specific infrared-dyed secondary anti-IgG antibodies (LI-COR Biosciences, Lincoln, NE, USA) were used. For infrared detection, membranes were scanned in Odyssey Imager (LI-COR); the bands were quantified using the software Odyssey V3.0 (LI-COR).

Statistical analysis

All data are expressed as the mean±s.e.m. (n=7–12). Two-way analysis of variance (ANOVA) was used to determine the effects of different factors: diet (control or cafeteria) and feeding conditions (fasting versus ad libitum feeding). Single comparisons were assessed by Student’s t-test. Correlations were assessed by Pearson’s correlation coefficients. The analyses were performed with SPSS for Windows (SPSS, Chicago, IL, USA). Threshold of significance was defined at P<0.05.

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Results

Body weight, food intake and blood parameters

Total food intake consumed by ad libitum-fed animals was measured. In the case of cafeteria diet-fed animals, the exact amount of each food item eaten was recorded, because not all the components of the diet are eaten equally.21 Notably, cafeteria animals ate three times the amount of kcal eaten by control animals (P<0.05, Student’s t-test; Table 1). The cafeteria diet, calculated based on the foodstuffs ingested by the animals, provided 40.3% of energy from carbohydrates, 46.3% from fat and 13.4% from proteins, whereas control diet provided 73% of energy from carbohydrates, 8% from fat and 19% from proteins. As shown in Table 1, although 12 days of cafeteria diet did not produce any significant changes in body weight, pups fed on a cafeteria diet presented greater body fat content than controls, as well as a greater size of the main WAT depots (inguinal, mesenteric, retroperitoneal and edipidymal), and lower liver weight. Notably, no correlation was found between body fat content and body weight, evidencing that increased adiposity is independent of increase in body weight. Animals in fasting conditions presented lower body weight, lower body fat content and lower liver weight compared with ad libitum-fed animals. Notably, the reduction of body fat content and liver size was greater in controls than in cafeteria-fed animals (P<0.05, interactive effect between diet and fasting, two-way ANOVA). Moreover, in control animals, but not in cafeteria animals, the size of the inguinal, retroperitoneal and epididymal WAT was significantly lower in fasted animals as compared with the ad libitum-fed ones (P<0.05, Student’s t-test).


Circulating levels of leptin, triglycerides and free fatty acids were higher in cafeteria-fed animals when compared with controls (P<0.05, effect of diet, two-way ANOVA). Glucose levels were lower in cafeteria animals, but only under ad libitum feeding conditions (P<0.05, Student’s t-test). Control and cafeteria animals under fasting conditions displayed lower circulating glucose and leptin levels (P<0.05, effect of fasting, two-way ANOVA). No changes were found regarding circulating insulin levels. Notably, under ad libitum feeding conditions, circulating leptin levels positively correlates with body fat content (r=0.850, P<0.001) but not with body weight (r=0.011, P=0.960).

Expression of genes related with energy balance in the hypothalamus

The mRNA expression levels of Insr, Lepr, Ghsr, Socs3, Stat3, Npy and Pomc in hypothalamus are shown in Figure 1. Ghsr expression levels were reduced in cafeteria-fed animals when compared with controls (P<0.05, effect of diet, two-way ANOVA), whereas no differences were observed either for Insr or Lepr expression levels. The mRNA levels of the aforementioned receptors remained unchanged under fasting conditions compared with ad libitum-fed conditions. The expression of Socs3 was lower in fasted animals compared with their ad libitum counterparts (P<0.05, effect of fasting, two-way ANOVA), especially in control animals (P<0.05, Student’s t-test). A similar trend was also observed in Stat3 mRNA expression (P=0.60, effect of fasting, two-way ANOVA). Animals fed on a cafeteria diet presented lower Npy mRNA levels than controls under both ad libitum feeding and fasting conditions (P<0.05, effect of diet, two-way ANOVA). No differences regarding Pomc expression were found. Notably, control animals displayed a higher Npy/Pomc mRNA ratio than cafeteria animals, particularly under fasting conditions; however, the fasting-induced increase in the aforementioned ratio was higher and significant by Student’s t-test only in control animals.

Figure 1.
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Expression of selected genes related with energy metabolism in the hypothalamus of 22-day-old male rats fed a control or a cafeteria diet from day 10 of life and killed under ad libitum feeding conditions and after 12h fasting. Data are mean±s.e.m. (n=10–13). Genes determined were: InsR, Lepr, ghrelin receptor (Ghsr), the SOCS3, Signal transducer and activator of transcription 3 (Stat3), Npy and Pomc. Statistics: F, effect of feeding condition (ad libitum/fasting); D, effect of diet (control/cafeteria), (P<0.05, two-way ANOVA). #, cafeteria versus control diet (P<0.05, Student’s t-test). *, fasting versus ad libitum feeding conditions (P<0.05, Student’s t-test).

Full figure and legend (111K)

Expression of genes related with energy metabolism in liver

Results showing the expression of selected genes involved in nutrient handling and metabolism in liver are summarized in Figure 2.

Figure 2.
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Expression of selected genes related with energy metabolism (fatty acid uptake, glucose uptake and metabolism, lipogenesis, lipid droplet physiology, lipolysis and fatty acid oxidation and leptin and insulin signaling) in the rpWAT of 22-day-old male rats fed a control or a cafeteria diet from day 10 of life and sacrificed under ad libitum feeding conditions and after 12h fasting. Data are mean±s.e.m. (n=10–13). Genes determined were: Cd36, lipoprotein lipase (Lpl), solute carrier family 2 (facilitated glucose transporter), member 4 (Slc2a4), hexoquinase II (Hk2), peroxisome proliferator activated receptor gamma (Pparg), Srebf1, Fasn, Cell death activator CIDE-A (Cidea), patatin-like phospholipase domain containing 2 (Pnpla2), carnitine palmitoyl transferase 1b, muscle (Cpt1b), leptin (Lep) and Insr. Statistics: F, effect of feeding conditions (ad libitum/fasting); D, effect of diet (control/cafeteria); DxF, interactive effect between diet and feeding condition, (P<0.05, two-way ANOVA). #, cafeteria versus control diet (P<0.05, Student’s t-test). *, fasting versus ad libitum feeding conditions (P<0.05, Student’s t-test).

Full figure and legend (154K)

Control animals fed ad libitum presented higher expression levels of Cd36 in liver than cafeteria rats (P<0.05, Student’s t-test). Cd36 mRNA levels tended to decrease in controls but increase in cafeteria animals under fasting conditions (P<0.05, interactive effect between diet and fasting, two-way ANOVA). The expression of lipogenic genes (Srebf1, Fasn and Scd1) and glucokinase was lower in animals under fasting compared with ad libitum feeding conditions in both control and cafeteria groups, although controls, compared with cafeteria animals, showed a greater expression of these genes under ad libitum feeding conditions (P<0.05, interactive effect between diet and fasting, two-way ANOVA). In fact, the fasting/fed mRNA expression ratio of Sreb1f, Fasn and Scd1 was higher in the cafeteria group (0.43, 0.22 and 0.08, respectively) than in the control group (0.06, 0.08 and 0.03, respectively). In turn, cafeteria animals displayed in the fasting state, higher mRNA expression levels of genes related to fatty acid oxidation: Ppara, Cpt1a and Pdk4 (P<0.05, interactive effect between diet and fasting, two-way ANOVA). Regarding the expression of genes related to leptin and insulin signaling, cafeteria-fed animals exhibited a greater mRNA expression of leptin and Insr (P<0.05, effect of diet, two-way ANOVA). Fasting increased Lepr mRNA expression in cafeteria animals, whereas no effect was observed in controls (P<0.05, Student’s t-test), and decreased mRNA levels of Socs3 in both groups (but only significantly by Student’s t-test in cafeteria-fed animals) and of Irs1 only in control animals (P<0.05, interactive effect between diet and fasting, two-way ANOVA).

In view of the differences found in the expression of genes related with insulin signaling, we analyzed protein levels of InsR and AKT (a downstream protein in the cascade of insulin signaling) by western blot. Results are shown in Figure 3. Notably, as occurring with mRNA expression levels, cafeteria animals also displayed greater abundance of the InsR protein compared with controls (P<0.05, effect of diet, two-way ANOVA). In addition, InsR levels were found to be higher in both control and cafeteria groups under fasting conditions (P<0.05, effect of fasting, two-way ANOVA), although the increase was more marked and significant by Student’s t-test in the control group (P<0.05). Regarding AKT, no significant differences were found between cafeteria and control animals; both groups showed lower levels of this protein under fasting compared with ad libitum feeding conditions (P<0.05, effect of fasting, two-way ANOVA).

Figure 3.
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InsR, AKT, AMPK and phosphoAMPK (pAMPK) protein levels (measured by western blotting) in liver of 22-day-old male rats fed a control or a cafeteria diet from day 10 of life and killed under ad libitum feeding conditions and after 12h fasting. Results represent mean±s.e.m. (n=10–13) expressed in arbitrary units (AU) and corrected by β-actin. Representative results are shown in the top left panel.

Full figure and legend (97K)

Knowing the role of the energy sensor AMP-activated protein Kinase (AMPK) in the regulation of cellular metabolism, we also analyzed its protein levels and its phosphorylated active form, pAMPK. Control animals displayed lower AMPK protein levels under fasting conditions compared with ad libitum feeding, whereas in cafeteria animals, AMPK levels remained stable independently of feeding status (P<0.05, interactive effect between diet and fasting, two-way ANOVA). Furthermore, fasted animals in the cafeteria group presented higher amounts of pAMPK compared with controls (P<0.05, Student’s t-test). The effects of diet were more evident for the pAMPK/AMPK ratio, suggesting a greater activation of the AMPK pathway in cafeteria animals when compared with controls (P<0.05, effect of diet, two-way ANOVA).

Expression of genes related with energy metabolism in rpWAT

The retroperitoneal fat pad was chosen as representative of WAT because of its relationship with the development of insulin resistance and type 2 diabetes.22 Results showing the expression of selected genes involved in nutrient handling and metabolism in this fat depot are summarized in Figure 4.

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Expression of selected genes related with energy metabolism (fatty acid uptake, lipogenesis, fatty acid oxidation, glucose metabolism and leptin and insulin signaling) in liver of 22-day-old male fed a control or a cafeteria diet from day 10 of life and killed under ad libitum feeding conditions and after 12h fasting. Data are mean±s.e.m. (n=10–13). Genes determined were: Cd36, Srebf1, Fasn, Scd1, peroxisome proliferator activated receptor alfa (Ppara), Cpt1a, Pdk4, glucokinase (Gck), Lepr, the Socs3, Insr and insulin receptor substrate 1 (Irs1). Statistics: F, effect of feeding condition (ad libitum/fasting); D, effect of diet (control/cafeteria); DxF, interactive effect between diet and feeding condition, (P<0.05, two-way ANOVA). #, cafeteria versus control diet (P<0.05, Student’s t-test). *, fasting versus ad libitum feeding conditions (P<0.05, Student’s t-test).

Full figure and legend (158K)

Animals under fasting conditions presented higher mRNA levels of lipoprotein lipase compared with those under ad litibum feeding (P<0.05, effect of fasting, two-way ANOVA); this difference was especially manifested in cafeteria-fed animals (P<0.05, Student’s t-test). No differences between groups were found in Cd36 mRNA expression. As a general trend, an interactive effect between the type of diet and feeding conditions was found in the genes studied related with glucose uptake and metabolism, lipogenesis, Cidea (which is involved in lipid droplet physiology) and Insr. Specifically, control animals under ad libitum feeding conditions displayed a higher expression of Slc2a4 and Hk2 compared with cafeteria-fed animals; these animals also showed a greater decrease in their expression levels under fasting conditions (P<0.05, interactive effect between diet and fasting, two-way ANOVA). In addition, control animals displayed a marked decrease in the expression levels of Pparg, Srebf1 and Cidea under fasting conditions compared with ad libitum conditions, whereas the expression levels of these genes in cafeteria animals were not affected (or not significantly in the case of Pparg), by fasting conditions. Control animals fed ad libitum exhibited a greater expression of Fasn compared with cafeteria animals (P<0.05, Student’s t-test), whereas no differences were found between groups under fasting conditions. Furthermore, the expression levels of this gene decreased as an effect of fasting in both groups, but the percentage decrease was more prominent in control animals. Regarding Insr, controls but not cafeteria animals, showed increased mRNA expression levels of this gene under fasting conditions, compared with those under ad libitum feeding (P<0.05, Student’s t-test), and hence fasted cafeteria animals displayed lower expression levels than fasted control animals (P<0.05, Student’s t-test). In addition, cafeteria-fed animals presented a lower expression of Pnpla2 and higher of Cpt1b compared with control animals (P<0.05, effect of diet, two-way ANOVA). Finally, as reflected in the circulating leptin and related to the greater amount of fat, cafeteria animals presented higher expression levels of leptin in WAT than control animals (P<0.05, effect of diet, two-way ANOVA); both groups decreased their expression under fasting conditions (P<0.05, effect of fasting, two-way ANOVA).

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Discussion

This study aimed to examine the influence of a short period of exposure to a cafeteria diet in early infancy in rat pups on the molecular adaptation to fed/fasting conditions in key metabolic organs, to gain some insight into the initial molecular events that may determine later outcomes of this dietary challenge. For this purpose, we introduced a cafeteria diet to 10-day-old male pups, around the date of natural transition from sole reliance on mother’s milk to the onset of solid food intake,23 and analyzed on postnatal day 22 the metabolic status of these animals under feeding and fasting states. We chose the cafeteria diet because it has been proven as an interesting tool to induce voluntary hyperphagia and hence overweight/obesity in rats and better resembles the Western diet in humans.6, 7, 8 Interestingly, we observed here an exacerbated accumulation of fat in the absence of notorious body weight increase, an effect not previously described so far that may have gone unnoticed, and that may be particularly harmful at young ages for later metabolic health.

The intake of a short period (12 days) of a cafeteria diet in 10-day-old pups induced hyperphagia and hence the intake of excess calories, 46% of which came from fat. Hyperphagia was accompanied by a marked increase in fat mass deposition, as in this short period, cafeteria animals had almost double the fat mass of control rats. Accumulation of excess fat during infancy is of particular concern, as early rapid growth and early adiposity rebound in humans have been proposed as probable early markers of adult obesity.24 In addition, several animal studies show that maternal exposure to a ‘junk’ food diet or to a cafeteria diet during pregnancy and lactation irreversibly promote adiposity in offspring, even when those offspring were fed exclusively a balanced chow diet after weaning.25,26 Moreover, feeding behavior is also affected by early exposure to ‘junk’ food, favoring the taste for this kind of food,27 or producing long lasting structural changes affecting satiety mechanisms.28 In addition to the hyperphagia observed in cafeteria-fed animals, the unbalanced composition of the diet (high in fat and low in protein) could also account for the overall effects observed here.

The hypothalamus–adipose axis highly contributes to food intake control. Leptin has a pivotal role in this regulation, because it is secreted mainly by the adipose tissue in proportion to the amount of fat stores and targets its hypothalamic receptor by increasing the expression of anorexigenic neuropeptides and inhibiting the expression of oxygenic ones, particularly Npy.29 Cafeteria-fed animals presented higher circulating leptin levels, reflecting larger fat depots and lower hypothalamic Npy expression (associated with higher leptin signaling) compared with control animals. However, the greater calorie intake observed in these animals is far beyond their metabolic needs, and represents a clear dysfunction in the control of food intake and energy balance. The failure of this hyperleptinemia to produce any compensatory decrease in food intake may be interpreted as evidence of leptin resistance.30 In addition, in comparison with control animals, cafeteria animals showed an attenuated activation of the orexigenic pathway, for example, the Npy/Pomc mRNA ratio, after 12h fasting, also indicating an impaired response to fed/fasting conditions.

Although cafeteria-fed animals did not display significant changes in circulating insulin levels compared with their controls, they seem to present other alterations related with insulin signaling at peripheral level, particularly in WAT. Insulin signaling in WAT enhances lipid storage by both stimulating triacylglycerol synthesis and inhibiting its breakdown. During fasting, the presence of lower insulin levels helps to inhibit lipogenesis and increase lipolysis.15 Notably, under fasting conditions, cafeteria animals did no decrease the expression of key transcriptional factors related with lipogenesis, Pparg and Srebf1, despite being under a situation of energy demand, indicating an impaired response to fed/fasting conditions. In addition, the reduced lipolytic capacity found in cafeteria animals could explain the lower decrease in the size of fat stores that these animals show under fasting conditions compared with controls. Active hydrolysis of stored triacylglycerols occurs during starvation through the actions of lipases (such as ATGL) and other regulators localized on lipid droplets. For example, several proteins associated to lipid droplets are known to be involved in lipid droplet physiology.31 Among them, CIDEA has emerged as an energy homeostasis regulator.32 CIDEA localizes lipid droplets and regulates their enlargement, thereby restricting lipolysis and favouring storage. CIDEA expression is controlled by PPARγ. PPARγ agonists upregulate Cidea expression, hence increasing lipid deposition. A depletion of CIDEA markedly elevates lipolysis in human adipocytes.33 In turn, Cidea-null mice exhibit a lean phenotype, increased lipolysis and resistance to high-fat diet-induced obesity and diabetes.32 Interestingly, here we observed that fasting produced a decrease in Cidea mRNA expression in control animals, which may favor increased lipolysis, but not in cafeteria animals. This impaired response to feeding conditions, together with the lower expression levels of ATGL, the main lipase involved in lipid mobilization, found in cafeteria animals compared with controls suggest that these animals exhibit an impaired ability to mobilize lipid stores. These early signs of metabolic disturbances occurring in WAT of cafeteria-fed pups may contribute to the exacerbated accumulation of fat already occurring at this early age and appears to be of huge relevance in later obesity related pathologies. Other adaptations occurring in WAT of cafeteria animals, such as decreased expression levels of Slc2a4, Hk2 and Fasn, and the increased expression of Cpt1b, may be interpreted as adaptations to the greater fat content of this diet in comparison to the control diet.

The AMPK is the principal internal cell-energy sensor.34 As a general trend, AMPK switches off synthesis and storage of fat and promotes fatty acid oxidation to generate ATP.35,36 High-fat diet-induced obesity in rats has been generally associated with a decrease in AMPK activity in multiple tissues.37 Conversely, here we found that cafeteria-fed animals presented higher hepatic pAMPK/AMPK ratio than control rats, both under ad libitum feeding and fasting conditions, suggesting a greater activation of the AMPK pathway. These different results could be tentatively explained by differences in the time and duration of the dietary challenge, and in the disturbances produced in leptin sensitivity, which may affect the leptin–AMPK axis. Leptin has been described to increase AMPK pathway, both in muscle and liver.38, 39, 40 Therefore, increased plasma leptin levels, together with increased mRNA levels of the Lepr in liver, which occur in cafeteria animals, could be responsible for the increased activation of the AMPK pathway seen in these animals, regardless of the presence of initial hallmarks of leptin resistance in other tissues, particularly at hypothalamic level. Fatty acid metabolism pathway is one of the best-characterized downstream targets of AMPK.41 AMPK may downregulate the expression of enzymes involved in fatty acid synthesis at transcription level, probably via regulation of SREBP1c.42 Actually, we found that cafeteria animals presented a lower expression of lipogenic genes (Srebf1, Fasn and Scd1) under ad libitum feeding conditions, compared with control animals, and an exacerbated expression of genes related with fatty acid oxidation (Ppara, Cpt1a, Pdk4). Cafeteria-fed animals also presented increased expression levels of Insr and higher protein levels of InsR in liver. Insulin is known to directly regulate the expression of Srebp1c.43 Therefore, the decreased activation of Srebf1 expression, and consequently of its direct targets—such as Fasn and Scd1—under feeding conditions occurring in cafeteria animals compared with their controls, despite displaying increased abundance of InsR, could be tentatively interpreted as a failure in insulin signaling. Several studies in young animals showed that very high-fat diets produce a rapid deterioration in whole body insulin sensitivity (reviewed in Morrison et al.30), and this may influence the progression of insulin resistance leading to obesity and diabetes. However, whether insulin signaling in liver is already altered in our model is not clear, as no alterations were found in insulin levels or in hepatic AKT, which is involved in the insulin signaling pathway. All in all, these results suggest that a short period of a cafeteria diet feeding in young rats affects hepatic metabolism; the liver undergoes molecular adaptations apparently directed to maintain energy homeostasis under this dietary challenge, namely decreased expression of lipogenic related genes and increased expression of fatty acid oxidation-related genes, probably involving the activation of the leptin–AMPK axis.

In conclusion, we show here that the introduction of a cafeteria diet in early infancy in rat pups is enough to disturb the metabolic response to feeding and fasting conditions in key metabolic organs, particularly in WAT, in addition to inducing hyperphagia and exacerbated body fat accumulation, despite going unnoticed in terms of body weight.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This work was supported by the Spanish Government (AGL2012-33692), and the Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición, CIBERobn. Our Laboratory is a member of the European Research Network of Excellence NuGO (The European Nutrigenomics Organization, EU Contract: no. FP6-506360).

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