Associations between low birth weight and increased risk of metabolic syndrome have been described in several epidemiological studies. The thrifty phenotype hypothesis proposes that poor foetal and infant nutrition produce changes in glucose–insulin metabolism aimed to improve immediate survival, but which also result in greater risk of type 2 diabetes later in life (Hales & Barker 2001). Low birth weight can be caused by intrauterine growth restriction (IUGR) resulting from maternal malnutrition, cigarette smoking or pregnancy-induced hypertension among others (Bergmann et al. 2008). After birth, most of these infants show rapid weight gain, which confers them advantages in terms of survival, low stature prevention and decrease in the risk of subnormal intellectual development (Ong 2007). However, accumulating evidence also indicates that catch-up growth is an important component of programming adult diseases, and associations between early rapid growth and increased later body mass index (BMI) have been described in several cohort studies (Claris et al. 2010). Evidence from animal studies suggests that alterations in insulin signalling caused by IUGR affect the development of visceral fat depots, which are key in determining the subsequent metabolic phenotype (Morrison et al. 2010).
Strategies that promote growth while normalizing insulin sensitivity, favouring lean mass accretion and limiting visceral fat would benefit this population. The potential of functional ingredients such as pre- and probiotics on modulating the onset and development of metabolic disorders has been extensively reviewed (Cani & Delzenne 2009). Likewise, LC-PUFA and specially n-3 fatty acids have shown to modulate adipose tissue function, increasing lipid catabolism and decreasing lipogenesis in animal models (Kopecky et al. 2009). However, the programming effect of these ingredients administered early in life has not been tested.
The objective of the present study was to evaluate the effect of supplementation early in life (7–56 days) with a mixture containing long-chain polyunsaturated fatty acids (LC-PUFA, arachidonic acid and docosahexaenoic acid), a bovine milk-derived oligosaccharide (BMO) and a probiotic (Lactobacillus rhamnosus NCC4007) in the growth and metabolic status of IUGR rats.
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- Material and methods
- Conflict of interest
- Supporting Information
Supplementation with the mixture induced better growth during early life as indicated by the greater weight gain during lactation, greater femur length and a trend to have greater body length than IUGRc after the experimental diets. Similarly, improved growth recovery and restoration of intestinal permeability have been reported in neonatally stressed rats supplemented with a mixture containing arachidonic and docosahexaenoic acid, galacto- and fructo-oligosaccharides and Lactobacillus paracasei NCC2461 (Garcia-Rodenas et al. 2006). Better catch-up growth with the experimental mixture may have been the result of improved absorption mediated by GLP-2, which was increased in IUGRmx. GLP-2 is a key regulator of intestinal development and nutrient absorptive capacity (Sigalet 2012), and it increased intestinal glucose uptake in suckling pups when it was injected to their dams (Drozdowski et al. 2009). Similarly, others have reported that prebiotic treatment in obese mice increases intestinal production of GLP-2 (Cani et al. 2009), the mechanism of action may involve modulation of bacterial fermentation and increased production of short-chain fatty acids, which in turn stimulate GLP-2 production (Tappenden et al. 2003). Short-chain fatty acids have a trophic effect in the intestinal mucosa, which may explain the increased caecal weight observed in the present study and also reported in other experiments supplementing prebiotic oligosaccharides (Neyrinck et al. 2012).
Increased fat accumulation after catch-up growth is documented in low birth weight infants (Ibanez et al. 2008) and in animal models of IUGR (Bieswal et al. 2006, Bol et al. 2009, Isganaitis et al. 2009), but despite better growth, IUGRmx had less fat content than IUGRc at 52 days and was not significantly different from REF. Studies performed in a semistarvation-refeeding animal model indicate that the mechanism of catch-up fat includes a period of hyperinsulinaemia, decreased glucose utilization in the muscle and marked upregulation of insulin-mediated de novo lipogenesis (Cettour-Rose et al. 2005, Summermatter et al. 2009). In the present study, increased adiposity was observed in IUGRc males and females, but hyperinsulinaemia was only present in IUGRc males. A similar gender dimorphism has also been reported by others; male growth-restricted mice and sheep were more susceptible than females to develop insulin resistance and metabolic syndrome (Owens et al. 2007, Hermann et al. 2009). Like in this study, some animal and human interventions with pre- and probiotics have shown beneficial effects in obesity and adiposity: peri-natal supplementation with Lactobacillus rhamnosus prevented excessive weight gain during early life in infants (Luoto et al. 2010). Lactobacillus gasseri reduced mesenteric and subcutaneous adipose tissue in Zucker rats (Hamad et al. 2009) and abdominal visceral and subcutaneous fat in human subjects with high BMI (Kadooka et al. 2010). Lactobacillus paracasei caused reduction in body fat in mice fed a high-fat diet (Aronsson et al. 2010). Supplementation with oligofructose reduced body fat in high fat fed C57BL/6 male mice (Anastasovska et al. 2012), and long-chain inulin-reduced adiposity in female rats (Jamieson et al. 2008). Prebiotic fibres during growth (16 week from weaning) modulated weight gain afterwards in rats fed a high-fat diet (Maurer et al. 2010). LCPUFA have also been reported to affect body weight and adiposity; supplementation with arachidonic and docosohexaenoic acid for 8 week after weaning reduced epididymal fat pad weight in ApoE*3Leiden mice even after a period high-fat diet (Wielinga et al. 2012). Similarly, dietary n-3 LCPUFA from lactation until 42 days has been shown to reduce fat accumulation after a challenge with a western diet in a murine model (Oosting et al. 2010). But in the present study, a protective effect was not observed. Intra-uterine growth retardation is associated with development of non-alcoholic fatty liver disease in children (Nobili et al. 2007), and oral administration of probiotics has been reported to reduce high-fat diet-induced hepatic steatosis (Ma et al. 2008). In the present study, a decrease in hepatic TG was observed in IUGRmx males just after supplementation (day 58), but this effect was not persistent by 100 days.
After the challenge with the high-fat diet, there were no differences in energy intake, adiposity or glucose tolerance between the IUGRc and REF groups. This was unexpected as it has been reported that caloric restriction during intrauterine life promotes hyperphagia (Vickers et al. 2000) and amplifies the detrimental effect of high caloric intake in rats (Bieswal et al. 2006). The challenge in the present study differs from others in the shorter period length (42 days) and the composition of the diet (60% of dietary calories from fat, without added sucrose), and this may have affected the response.
The effects of maternal food restriction and mixture supplementation on hepatic gene expression at 58 and 100 days were gender-dependent. Sexual dimorphism in hepatic gene expression is regulated by growth hormone (GH) gender-specific secretion patterns (Clodfelter et al. 2007, Wauthier & Waxman 2008). GH deficiency caused major alterations in expression of genes involved in fatty acid, xenobiotic and steroid hormone metabolism (Amador-Noguez et al. 2005), which were functions affected by gender and treatment in the present study. Impairment of the somatotrophic axis may occur as a consequence of intrauterine undernutrition; IUGR newborn infants have higher cord blood GH (Setia et al. 2007), and children born small for gestational age have different GH pulse frequency and peak amplitude than those born with adequate birth weight (Woods et al. 2002). In rats, food restriction during lactation resulted in ‘female-like’ GH secretion patterns in the adult males, but no changes were seen in females (Houdijk et al. 2003). Interestingly, some of the genes in which IUGR caused expression changes in opposite direction between genders (Table 6) have been reported as having sex-specific modulation by growth hormone: hepatic expression of A1bg1, Akr1b7, Ifi47 and Prlr has been reported to decrease in females after hypophysectomy and respond to GH injection (Wauthier & Waxman 2008). In the present study, the expression of these genes was severely decreased in IUGRc females at 58 days, when they had significantly lower BW than reference. The extent of the differences in gene expression was reduced by 100 days when IUGRc females had completed catch-up growth, and therefore, their BW was not different from REF. Cyp2c11, whose expression has been shown to decrease in males after hypophysectomy (Wauthier & Waxman 2008), was severely decreased in IUGRc males at 100 days; moreover, female predominant genes (A1bg1, Akr1b7, Ifi47 and Prlr) were increased when compared with REF. In IUGRc males, BW remained numerically lower than REF through the study although the difference did not reach significance. These observations suggest that GH secretion pattern may be altered in the IUGRc group and could have caused sex-specific changes in hepatic gene expression, but we cannot be conclusive because GH pulses were not measured in the present study. Furthermore, the molecular mechanism by which the experimental mixture was able to normalize GH secretion, for example improving nutrient absorption, remains to be elucidated. Our DNA methylation analysis revealed a potential role of this molecular mechanism in the previously known gender-specific expression of Akr1b7 (Kotokorpi et al. 2004) and Ifi47 (Wauthier & Waxman 2008), and as well the known gender-specific and age-dependent expression of A1bg (Tiong et al. 2006). Nevertheless, our results indicate that DNA methylation is not underlying the treatment by gender interaction expression changes observed in the selected genes. Epigenetic mechanisms like the presence of DNase hypersensitive chromosomal regions and sex-specific chromatin organization also cause sexual differences in metabolic processes (Gabory et al. 2011) and may be responsible for the observed effects.
In conclusion, the mixture of bovine milk oligosaccharides, Lactobacillus rhamnosus NCC4007, arachidonic and docosahexaenoic acid improved catch-up growth and prevented excessive adiposity at short-term. Data from the microarrays outlined some interesting gender differences in response to maternal restriction and early nutritional intervention that suggest growth hormone regulation by IUGR. Our DNA methylation analysis indicated that this molecular mechanism could mediate some gender-specific and age-dependent gene expression patterns in the liver. After a period of high-fat diet, we did not observe any effect either from pre-natal restriction or from post-natal treatment.
The authors would like to thank Rodrigo Bibiloni, Florence Blancher, Christian Darimont, Lucie Deschamps, Patricia Leone, Taoufiq Harach, Massimo Marchesini, Christophe Maubert, Christine Mettraux, Mathieu Membrez, Kurt Ornstein, Stéphane Pinaud, Manuel Ribeiro, Isabelle Rochat, José Sanchez García and Marie Camille Zwalen.