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Obesity

Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

Effect of Antibiotic Treatment on Intestinal Microbial and Enzymatic Development in Postnatally Overfed Obese Rats Sˇtefan Mozesˇ, Zuzana Sˇefcı´kov a, Dobroslava Bujn akov a and Lubomı´r Racek

Objective: To investigate the effect of the microbiota-induced changes and early overfeeding after amoxicillin administration (a) in suckling pups via their dams up to 15 days of lactation and (b) in weaned pups on intestinal microbial/functional adaptability and obesity development in male Sprague-Dawley rats. Design and Methods: Postnatal nutrition was elicited by adjusting the number of pups in the nest to 4 (small litters [SLs]) and 10 (normal litters [NLs]), while from days 21 to 40, both groups were fed with a standard diet. The numbers of Bacteroides/Prevotella (BAC) and Lactobacillus/Enterococcus (LAB) in the jejunum and colon were determined by fluorescence in situ hybridization technique, and jejunal alkaline phosphatase (AP), a-glucosidase and aminopeptidase activity was assayed histochemically. Results: On day 40, the SL in comparison with NL animals displayed excess weight/fat gain accompanied by higher LAB and lower numbers of BAC, and with permanently higher AP activity. Moreover, these acquired changes continued in SL vs. NL rats and were not influenced by antibiotic treatment, which induced significant decrease in the quantity of LAB and BAC. Conclusions: These findings highlight the role of early life overfeeding upon the gut microbial/functional ontogeny and allow to distinguish their potential involvement in later risk of obesity. Obesity (2013) 21, 1635-1642. doi:10.1002/oby.20221

Introduction In general, obesity is characterized as a condition of altered energy balance control enabling more efficient energy harvesting and storage, accompanied by large bacterial community variations in the digestive tract. The differences in quantity and proportion of two dominant divisions of gut microbiota, namely the increased numbers of Firmicutes and reduction of Bacteroidetes, have been demonstrated in genetically obese animals and in humans (1,2), suggesting that gut microbiota could be a contributing factor to obesity. However, at present time, the question of a possible relationship between the gut microbiota and obesity in humans is highly complex and remains unclear (3). Regarding the Firmicutes, there were found no differences between obese and nonobese adult individuals, and in obese in comparison with lean peoples even a significantly lower proportion of Firmicutes and increased proportion of Bacteroidetes has been documented (4,5). Altogether, these data have not revealed any causative link between gut microbiota variations and obesity status; however, due to the existence of several host-related environmental factors (age and dietary conditions), these influences might also be considered as a part of a mechanism that additionally affects the gut microbiota composition and metabolic homeostasis.

The microbial colonization of the immature gastrointestinal system is considered as a critical event in programming the future physiological processes involved in energy balance control. Accordingly, great changes in gut microbiota have been reported in breast-fed preweaned infants (6,7) as well as suckling mice (8) and rats (9,10). There is also evidence that gut microbial colonization in germ-free rodents itself markedly decreased the level and activity of the small intestinal digestive and absorptive enzymes (alkaline phosphatase [AP] and disaccharidases) (11,12). Furthermore, data from another experiment in which overfeeding in rat pups was elicited by postnatal reduction of the litter size (leading to excessive maternal milk fat production) revealed that pre-weaning nutritional events are particularly critical for developmental mutuality between obesity-promoting gut microbiota and increased AP activity (13). At the present time relatively little information is available about the impact of microbiota destabilization on intestinal enzymatic activity during the suckling and weaning periods. It has been documented that antibiotics administration suppressed the numbers of aerobic and anaerobic bacteria in the small and large intestines in suckling nonobese rats (14) and in adult ob/ob mice (15). One report also showed the increased activity of sucrase but unchanged activity of lactase and maltase in the small intestine of suckling rat pups

Department of Developmental Physiology, Institute of Animal Physiology, Slovak Academy of Sciences, Sˇoltesovej 4-6, 040 01 Kosˇice, Slovak Republic. Correspondence: Sˇtefan Mozesˇ ([email protected]) Disclosure: The authors declared no conflict of interest. Funding agency: This study was supported by grant 2/0019/11of the Slovak Academy of Sciences. Received: 2 April 2012 Accepted: 17 November 2012 Published online 12 December 2012. doi:10.1002/oby.20221

www.obesityjournal.org

Obesity | VOLUME 21 | NUMBER 8 | AUGUST 2013

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Antibiotics, Gut Development, Obesity Mozesˇ et al.

influenced via maternal antibiotic treatment (16). However, information about the effect of similar experimental interventions on the ontogeny of digestive enzymes in obese suckling rat pups is lacking. Moreover, although it has been found that antibiotic treatment-associated changes in gut microbiota in adult mice improved high-fat diet-induced metabolic disorders (diabetes, endotoxemia, and overweight), these changes did not affect the elevated energy intake and only partially normalized the abundant body fat accumulation in these animals (17). In this sense, comprehensive data about the effect of the gut microbiota and early life overfeeding throughout different periods of life in obese vs. lean animals may highlight their specific role in energy homeostasis adjustment and allow to distinguish their potential involvement in later risk of obesity. To test this hypothesis, we investigated the consequences of postnatal as well as post-weaning antibiotic treatment in rat pups nursed either in reduced or in normal litters (NLs). In this report, we examined the differences in the number of two microbial groups, i.e. Lactobacillus/Enterococcus (members of the Firmicutes) and Bacteroides/Prevotella (members of the Bacteroidetes) in the small and the large intestine as well as the differences in brush border-bound AP, a-glucosidase, and aminopeptidase activity in the jejunum in SL vs. NL 40 days old rats.

Methods and Procedures Animals and experimental protocol Sprague-Dawley virgin rat dams (Charles River Laboratories, Prague, Czech Republic), weighing 240-260 g, mated at 10 weeks of age were used in this study. The rats were individually housed in Plexiglas cages and kept under conditions of constant room temperature (22 6 2 C), relative humidity (55 6 10%), and 12 h light/dark cycle (light on 0600 to 1800 h) and had free access to a standard laboratory diet (Laboratory diet M1, Ricmanice, Czech Republic; containing 13.4 kJ/g, with 26.3% energy as protein, 9.5% as fat, and 64.2% as carbohydrate) and tap water. Within 24 h of parturition, the rat dams with less than 8 or more than 12 pups in the nests were excluded from the experiment. To induce normal or overnutrition in their offspring, the litter size in one group of dams (n ¼ 4) was adjusted after parturition to 10 pups per nest (NL) and in the other group of dams (n ¼ 8) to 4 pups per nests (small litters [SLs]). Animals arising from these adjusted nests (consisting of males and females) were submitted to different antibiotic intake procedures during suckling in comparison with the post-weaning period. In the first part of the experiment, dams nursing pups in normal and in reduced nests were randomized into two groups after parturition: (a) experimental dams receiving Amoxygal plv.sol (amoxicillinum trihydricum; Pharmagal, Nitra, Slovak Republic) in their drinking water up to 15 days of lactation and (b) the control dams drinking only tap water. Fresh antibiotic solution was prepared twice daily (30 mg/kg and concentration 0.2 g/l). In the second part of the experiment, 21-day-old-male pups of each group (n ¼ 16) were housed individually under the same conditions (diet, temperature, and light dark regime) as before weaning with the antibiotic intervention (induced via drinking water) that was continued from weaning to day 40 for only eight rats per group. To determine the growth and post-weaning feeding performance in these animals, the body weight was measured every 5 days, and from day 21 until day 40 their food intake was recorded at 48-h intervals. On day 40, samples of adipose tissues (epididymal and

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perirenal adipose depots) and jejunal samples (at one-third of the intestinal length) for enzyme assay and for enumeration of bacteria were taken after decapitation of the animals (from 0800 to 0900 h).

Ethics statement All animal experiments were reviewed and approved by the Ethical Committee for animal experimentation of the Institute of Animal Physiology, approved by the State Veterinary and Food Administration of the Slovak Republic, and were performed in accordance with Slovak legislation 23/2009 on the protection of animals used for experimental and other scientific purposes.

Fluorescence in situ hybridization Fresh jejunal samples were sectioned into small pieces, cut longitudinally, washed thoroughly with sterile phosphate-buffered saline (pH 7.4) and the jejunal mucosa was removed for homogenization. Homogenized mucosa was fixed in 4% paraformaldehyde (Fluka, Buchs, Switzerland) overnight at 4 C and then stored in equal volumes of phosphate-buffered saline and 96% ethanol at -20 C. A similar procedure was used in collection and preparation of luminal colonic samples. The numbers of intestinal microbial communities in the jejunum and colon were assessed using the fluorescence in situ hybridization method with probes (VBC-Genomics, Austria) Lab158 for Lactobacillus/Enterococcus sp. group Cy3—5’-GGT ATT AGC A(C/T)C TGT TTC CA-3’ (18) or Bac303 for Bacteroides/Prevotella group FITC— 5’-CCA ATG TGG GGG ACC TT-3’ (19). Aliquot volume of the fixed jejunal mucosa or colonic content was added to 100 ll permeabilization solution Tris/HCl buffer (10 mM Tris and 1 mM EDTA) at pH 6.5 with 100 mg/ml lysozyme and treated 1 h at 37 C. Permeabilized samples were mixed with hybridization solution (900 mM NaCl, 20 mM Tris–HCl, pH 8.0, and 0.01% sodium dodecyl sulfate), contained a probe (0.5 pmol/ll) and placed into a hybridization apparatus at appropriate temperatures overnight (Bac303 at 46 C and Lab158 at 50 C). The hybridized samples were vacuum filtered onto 0.2-lm polycarbonate membrane filters. A microscope (Olympus, Tokyo, Japan; BX 51) fitted with appropriate filters for Cy3 dye and FITC dye was used for the enumeration of bacteria. A minimum of 20 fields were counted for each filters. The number of bacteria was calculated from formula: number of bacteria/gram of samples ¼ X M Df/S, where X is the number of positive bacteria per field of view; M the total number of fields per effective filter surface different for each microscope and magnification used; Df the dilution factor; and S the weight amount of samples in grams. Values of microbiota in jejunal and colonic samples are given as log (numbers of bacteria/ 0.1 g jejunal mucosa or 0.1 g colonic content).

Enzyme assays For enzyme assay, small (0.5 cm) segments of the jejunum were immediately removed, the lumen was rinsed in distilled water and frozen in liquid nitrogen. Segments of the frozen tissue were cut (8 lm) in a cryostat at -25 C and the tissue slices were transferred to glass slides and air dried. The analysis of AP activity was performed using a modified simultaneous azocoupling method (20). The incubation medium contained 2.0 mmol/l naphthol AS-BI phosphate (Sigma, Deisenhofen, Germany), 0.8 mmol/l hexazotized new fuchsin (Serva, Heidelberg, Germany), and 0.05 mol/l veronal acetate buffer. The sections were incubated at 37 C for 10 min at pH 8.9


Original Article

Obesity

OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY

TABLE 1 Effect of maternal antibiotic treatment on somatic and intestinal parameters in 40-day-old SL and NL pups

Control NL Body weight (g) Weight gain (g) (day 1–40) Fat pads weight (% b.w.) Alkaline phosphatase (AP) a-Glucosidase AMP Lactobacillus/Enterococcus jejunum Bacteroides/Prevotella jejunum Lactobacillus/Enterococcus colon Bacteroides/Prevotella colon

155.4 148.4 0.50 12.0 13.7 19.8 7.40 8.06 8.00 8.44

6 6 6 6 6 6 6 6 6 6

5.9 5.9 0.05 0.4 0.5 0.9 0.02 0.05 0.05 0.13

Antibiotic NL 163.3 156.8 0.54 12.5 14.9 20.5 6.43 6.16 7.54 7.23

6 6 6 6 6 6 6 6 6 6

6.3 6.1 0.04 0.5 0.6 0.9 0.16*** 0.11*** 0.08*** 0.06***

Control SL 185.3 178.7 0.78 14.8 15.4 19.8 8.19 7.15 9.10 7.65

6 6 6 6 6 6 6 6 6 6

a

4.8 4.8a 0.06a 0.4c 0.5 0.7 0.09c 0.06c 0.05c 0.10c

Antibiotic SL 188.0 181.3 0.78 14.2 15.1 21.9 6.93 6.71 8.40 7.47

6 6 6 6 6 6 6 6 6 6

5.4b 5.3b 0.04a 0.4b 0.6 0.5 0.04**c 0.12*c 0.05***c 0.05

Values are expressed as means 6 SEM (eight animals/group). Brush border-bound jejunal enzyme activity is given as density values (pixel intensities) at wavelength 520 nm. Values of microbiota in jejunal and colonic samples are given as log (numbers of bacteria/0.1 g jejunal mucosa or 0.1 g colonic content). Significantly different from control group *P < 0.05, **P < 0.01, ***P < 0.001. Significant differences among NL and SL groups a P < 0.001; b P < 0.01; c P < 0.0001 by Tukey’s multiple comparison test after two-way ANOVA.

(21). Demonstration of a-glucosidase (maltase glucoamylase complex EC 3.2.1.20) activity was performed using the simultaneous azocoupling method (20) with 2-naphthyl-a-D-glucopyranoside as substrate, hexazotized new fuchsin, N,N-dimethylformamide and 0.1 M citric acid–phosphate buffer. The sections were incubated at 37 C and pH 6.5 for 20 min. The analysis of aminopeptidase M activity was performed using the simultaneous azocoupling method (22) with L-leucyl-4-methoxy-2-naphthylamide as a substrate, Fast Blue B, N,N-dimethylformamide and 0.1 M citric acid–phosphate buffer. The sections were incubated at 37 C for 6 min at pH 7.0. The histochemically stained slides were illuminated with white light after filtering with a 520-nm monochromatic filter and visualized by image analysis performed by the Ellipse program (ViDiTo, Slovakia) where the gray level of each pixel was given by a value in the 0-255 range. The correspondence between these gray level values and the known integrated absorbance values of the same section points was determined by calibration. A special semi-interactive algorithm was used to find relevant pixels along the villus length whose density was measured (23). Quantification of the enzyme activity (pixel intensities) was performed along the villus length in a whole section of at least four samples, and the mean values recorded were referred to one animal.

Statistical analysis Statistical analyses were performed using the software package Statistica AXAZ (StatSoftCR, Czech Republic). Two-way ANOVA and Tukey’s post hoc test were used for detecting significant differences between SL and NL groups and for evaluating their responses to antibiotic treatment. Data were expressed as means 6 SEM and statistical significance was accepted at P < 0.05 level.

Results Litter size manipulations by adjusting the number of pups in the nest to 4 (SL) or to 10 (NL) were accompanied by the appearance


of profound somatic changes characterized by significantly higher mean body (Tables 1 and 2) and fat pad weights in SL pups (Figure 2A and B). Moreover, these acquired differences persisted until day 40 and were not affected by their change to normal diet consumption after weaning. Litter size differences also significantly influenced the intestinal microbial/enzymatic maturation during the entire experiment. In SL control rats, higher numbers of Lactobacillus/Enterococcus group (LAB) and reduced numbers of Bacteroides/Prevotella group (BAC) in the gut were discovered, whereas NL controls are characterized by lower LAB and elevated BAC numbers in the jejunum and colon on day 40. The imprint of litter size manipulation was also accompanied by distinct AP activity adjustment, i.e. in SL when compared with NL controls significantly higher AP activity was recorded on day 40. However, under these conditions, a similar association between altered somatic/metabolic homeostasis and changes in other brush border enzyme activity was not established. Activity of a-glucosidase and aminopeptidase during suckling and post-weaning periods in control SL vs. NL animals did not show any significant changes (Tables 1 and 2). The presented results show that antibiotic administration substantially altered the microbial colonization in SL and NL pups. Accordingly, the maternal amoxicillin treatment (until day 15) resulted in significantly reduced LAB and BAC numbers even in 40-day old animals (Table 1). Regarding the efficiency of microbial manipulation (induced via drinking water) during the post-weaning period, the presented results reveal the sufficient adaptability of gut microbiota to this antibiotic challenge. In comparison with controls, the antibiotic-treated animals showed a distinct reduction in their LAB and BAC parameters (Table 2). However, under these conditions, no parallel antibiotic effect was seen either on body weight, fat pads, or enzymatic parameters. In SL pups, activity of jejunal AP remained significantly higher on day 40 than in NL animals (Table 1). This tendency was also established in post-weaning antibiotic-treated SL and NL rats, i.e. the depression of gut microbiota development did not influence their previously acquired intestinal enzyme diversity and obese or lean status (Table 2).


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TABLE 2 Somatic and intestinal parameters in 40-day-old SL and NL pups submitted to post-weaning antibiotic treatment

Control NL Body weight (g) Weight gain (g) (day 1–40) Fat pads weight (% b.w.) Alkaline phosphatase (AP) a-Glucosidase AMP Lactobacillus/Enterococcus jejunum Bacteroides/Prevotella jejunum Lactobacillus/Enterococcus colon Bacteroides/Prevotella colon

153.9 146.9 0.42 11.5 13.9 18.4 7.42 8.03 8.83 9.27

6 6 6 6 6 6 6 6 6 6

5.8 5.7 0.05 0.4 0.7 0.6 0.01 0.09 0.22 0.28

Antibiotic NL 160.9 153.9 0.53 11.7 14.0 19.3 6.49 6. 61 8.21 8.41

6 6 6 6 6 6 6 6 6 6

7.4 7.3 0.04 0.5 0.5 0.5 0.14*** 0.17*** 0.07* 0.02**

Control SL 178.4 171.9 0.79 13.8 14.8 19.0 8.38 7.43 10.29 8.50

6 6 6 6 6 6 6 6 6 6

a

3.2 3.1c 0.06a 0.4c 0.6 0.6 0.07b 0.07a 0.13b 0.03c

Antibiotic SL 183.6 177.1 0.72 13.4 14.5 19.1 6.84 6.80 8.22 8.22

6 6 6 6 6 6 6 6 6 6

3.1b 3.1c 0.05a 0.5c 0.5 0.6 0.03***c 0.11** 0.03*** 0.06

Values are expressed as means 6 SEM (eight animals/group). Brush border-bound jejunal enzyme activity is given as density values (pixel intensities) at wavelength 520 nm. Values of microbiota in jejunal and colonic samples are given as log (numbers of bacteria/0.1 g jejunal mucosa or 0.1 g colonic content). Significantly different from control group *P < 0.05, **P < 0.01, ***P < 0.001. Significant differences among NL and SL groups a P < 0.001, b P < 0.0001, c P < 0.01 by Tukey’s comparison test after two-way ANOVA.

Figure 1 shows a similar key role of early litter size manipulation on the food intake adjustment in pups submitted to antibiotic treatment during suckling (A) to that after the weaning period (B). Accordingly, despite the specific experimental design, during the post-weaning periods, the same differences were recorded, i.e. a significantly higher mean food intake (g/day) in SL control and SL antibiotic than in NL control and NL antibiotic animals.

Discussion Role of gut microbiota in obesity programming Our results provide new insights into questions concerning participation of gut microbiota in mechanisms that regulate somatic/metabolic homeostasis in obese SL vs. lean NL animals. From this point of view, the significant importance of the present results is the convergent evidence implicating the quantitative and proportional gut microbiota changes, i.e. reduced numbers of Bacteroides/Prevotella (members of the Bacteroidetes), higher numbers of Lactobacillus/ Enterococcus group (members of Firmicutes), as mediating factors of future obesity-prone and/or adverse processes. At the present time, however, the causal relationship between host metabolic efficiency and the distinctive characteristics of gut microbiota composition is poorly understood so their importance has to be considered with caution. Thus, our results revealed that the body weight, fat pads, and AP activity changes might rather be related to early overnutrition than to gut microbiota modification. Despite significantly reduced LAB and BAC numbers after antibiotic treatment, the control SL vs. the antibiotic SL group did not show any significant differences in body composition and AP activity on day 40. Regarding the complexity of the microbial ecosystem, there is also evidence from human and animal studies about the important role of other intestinal bacterial groups that may predict different metabolic phenotype development. In particular, the dominantly colonized Bifidobacteria in the gut (in comparison with the reduced proportion of Lactobacillus, Enterococcus, and Bacteroides groups) in breast milkfed infants is usually considered as a factor decreasing obesity risk (6,7); the numbers of Bifidobacteria are higher in normal weight,

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whereas the opposite tendency has been observed in overweight children and adult subjects (5,24). On the other hand, this finding was not supported in overweight adolescents submitted to dietary restriction. In these individuals, weight loss itself did not result in beneficial increase of Bifidobacteria numbers, and moreover their weight reduction significantly decreased number of Bifidobacterium longum (25).

Possible mechanisms of action of gut microbiota revealed by antibiotic treatment In comparison with humans (6), the natural gut colonization in mice is associated with more prominent increase of Lactobacillus and Bacteroides than the Bifidobacteria densities during the suckling phase (8), and similar variability in gut microbiota is also manifested in mature rats (26). However, there exist several questions about the key role of gut bacteria regarding the development of obesity, since their overall involvement in processes regulating metabolism and body composition seems to be more plastic than was previously anticipated. Accordingly, it has also been documented that intestinal microbiota composition itself does not explicitly influence the energy balance equation. First, the results from an experiment in which dietary obese rats were submitted to consumption of high-fat diet supplemented with Bifidobacteria also revealed that such microbiota variations did not alter their obesity status (27). Second, our results demonstrate that experimental manipulations, i.e. quantitative reduction and proportional changes in gut microbiota after antibiotic treatment did not result in lower body fat gain in SL rats in comparison with the corresponding untreated SL animals. Finally, it has been shown that the absence of gut microbiota does not provide any general protection from diet-induced obesity, i.e. germ-free mice gained more weight and had more body fat when fed a high-fat diet than conventional mice (28).

Role of intestinal enzymes In general, the brush border-bound AP of intestinal enterocytes is considered as a representative enzyme that is functionally involved


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FIGURE 1 Mean food intake (g/day) from days 21 to 40 in control vs. pre-weaning antibiotic-treated (indirectly via the mother) (A) and in control vs. post-weaning antibiotic-treated (B) rats. Values are means 6 SEM (n ¼ 8 animals/ groups). Significant differences between NL and SL groups: *P < 0.05, **P < 0.01 by Tukey’s test after two-way ANOVA.

in nutrient/lipid absorption. It has been suggested that AP activity in rats displays circadian fluctuations closely related to food intake (29), and this activity markedly decreases after food deprivation


(30). Furthermore, increased AP activity occurs in response to increased fat intake, whereas under these conditions, no changes in other brush border enzyme activity were found (31,32). In the


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FIGURE 2 Epididymal and perirenal fat pads (% b.w.) in control vs. pre-weaning antibiotic-treated (indirectly via the mother) (A) and in control vs. post-weaning antibiotic-treated (B) rats. Values are means 6 SEM (n ¼ 8 animals/ groups). Significant differences between NL and SL groups: *P < 0.05, **P < 0.01, ***P < 0.01 by Tukey’s test after two-way ANOVA.

present study, we provide significant evidence that early fat-forced conditions facilitate AP activity expression in the jejunum of SL pups, and that this intestinal adaptability dominantly influenced their

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energy-harvesting efficiency. In accord with these data, in our previous study, we also documented that in suckling rat pups exposed to fat-rich milk, greater AP activity positively influenced their body


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weight and fat deposition throughout the pre- and post-weaning periods (33). Nevertheless, there also exists evidence of an inverse relation between intestinal AP and the rate of fat absorption in obese animals. In AP-deficient knockout mice after long-term exposure to a high-fat diet, accelerated fat absorption, and greater body weight and fat gain were observed in comparison with wild-type controls (34,35). Although the mechanisms underlying these disorders are as yet unclear, recent studies have revealed that AP-deficient mice had dramatically fewer and also different types of aerobic and anaerobic microbes in comparison with wild-type animals (36). However, the question regarding the role of dominant gut microbiota, and especially identification of which previously acquired host/microbe interactions can participate in the development of obesity in response to high-fat diet in these animals, needs to be explored in future studies.

Pre-weaning over-nutrition and development of obesity Regarding the early nutritional background, it has been documented that litter size reduction or feeding rat dams with high-fat/energy diet throughout lactation both increase the milk lipid concentration, and these changes positively correlate with excess fat deposition and weight gain in their offspring (10,13,37,38). Consistent with the animal studies, excess nutrition in lactating human mothers also enhances their milk energy/fat content as well as weight gain of their offspring between birth and 6 months of age (39). These findings support the concept that especially the early dietary practices might be considered as a positive risk factor for the development of obesity. Accordingly, our recent results show that change from overnutrition to standard diet from 15 to 20 days as well as from weaning to day 40 did not improve the previously acquired efficient energy-harvesting performance in obese animals. In addition, it was found that the number of obesity-related metabolic disorders (hyperphagia, hyperinsulinemia, elevated triglycerides and blood pressure, and overweight) in postnatally overfed SL rats persisted till their adult age (40). In conclusion, the presented data show that the initiation and maintenance of obesity is partially accompanied with gut microbial alterations in SL pups. Moreover, these comprehensive data indicate that the effect of the microbiota-induced changes might rather be considered as transitory, since obesity also persisted in antibiotic-treated pups. Since SL rats, which were overfed during neonatal life, displayed different maturation of digestive and absorptive enzymes, we might consider this early dietary imprint as a part of a mechanism that establish the future altered energy balance, body weight, and adiposity control. Since abundant food/energy intake is considered as the main cause of the worldwide increase in obesity, knowledge about the potential role of early-life dietary markers might be theoretically helpful and provide some perspective for better understanding of the mechanisms involved in the limited efficacy of several dietary manipulations/restrictions in overweight children and adults.O

Acknowledgment The authors thank Andrew Billingham for his revision of the English text. Sˇ.M.: conceived and designed the experiments; Z.Sˇ.,


L.R., D.B.: performed the experiments; Sˇ.M.: analyzed the data; and Sˇ.M., Z.Sˇ., D.B., L.R.: wrote the paper. C 2012 The Obesity Society V

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