As obesity rates escalate, it has become imperative to identify factors that contribute to the development of obesity and its comorbidities. The risk of developing the metabolic syndrome, a combination of risk factors predisposing individuals to cardiovascular and type 2 diabetes, has been shown to be particularly sensitive to nutritional influences early in life . Most animal studies have used two major models of metabolic programming, that of maternal undernutrition to probe prenatal influences , and litter size manipulation to examine postnatal under- and overnutrition during the suckling period . Much less work has examined the effects of maternal overnutrition and even less work has probed the effect of increased intake of select nutrients in the early postnatal period. Therefore, we examined the consequences of consuming a high prebiotic fiber or high protein diet throughout growth followed by a high energy diet challenge in adulthood.
The present study demonstrates that a long-term weaning diet high in fiber or protein results in significant differences in body weight and fat mass, the secretion of select satiety hormones, and the expression of genes involved in glucose and lipid metabolism in response to a high energy diet in adulthood. Our major findings include: 1) an increase in body weight and fat mass in response to high energy diets after a weaning diet high in protein compared to fiber; 2) an increase in energy intake in rats fed a HP diet, especially when switched to a HFHS diet; 3) a decrease in glucose and increase in GLP-1 in rats fed HF; and 4) greater accumulation of triglyceride in the liver of HP-fed rats. Take together, this data suggest that the response to a high energy diet challenge in adulthood results in greater metabolic dysfunction (ie. increased fat mass, decreased glucose tolerance, reduced GLP-1 secretion, greater hepatic triglyceride content) when a HP diet is consumed from weaning into early adulthood compared to a HF diet.
The increase in the relative mass of the small intestine, colon and cecum in the HF group is consistent with our previous work with fiber-enriched diets [10, 11, 24]. Addition of readily fermentable fiber to a diet is known to cause a significant proliferative effect in the colon and distal small intestine . The prebiotic fibers, inulin and oligofructose, are highly water soluble and non-viscous and stimulate lactic-acid bacteria growth in the gut . Inulin, chiefly derived from chicory, and its hydrolysis product, oligofructose, are fructans that have β-(2-1) linkages that differ in a high (inulin) and low (oligofructose) number of fructose molecules . Reductions in body weight and fat mass , enhanced satiety , improved glucose control in hyperglycaemic subjects , increased GLP-1 secretion  and improved blood lipid profiles have all been demonstrated in humans consuming oligofructose .
GLP-1 is released from L cells in the intestine in response to food ingestion and has been shown to slow gastric emptying and decrease food intake and body weight . We have previously shown that a diet high in fiber increases plasma GLP-1 concentrations [10, 11, 24]. There is some controversy surrounding the role of anorexigenic hormones, including GLP-1, in enhancing satiety with HP diets . This study demonstrates that HF caused a significant sustained release of GLP-1 which was not observed in the HP or C rats. This increase is in agreement with work by others feeding oligofructose [40–42]. Similarly, amylin, a pancreatic β-cell hormone that inhibits food intake  was also increased by the HF diet in females. No significant differences between groups were seen in males which is likely due to the large variability observed in the males. Our observations of increased amylin in the HF-fed female rats is in agreement with that seen by Cani et al.  wherein consumption of the prebiotic fiber, oligofructose, increased amylin in ob/ob mice. The change in these two anorexigenic hormones lends support for the lower energy intake observed in the HF rats both in periods of direct exposure to the prebiotic but also in the period where all rats consumed the HFHS diet. The increase in body weight and energy intake was greatest in HP rats when they were introduced to the HFHS diet suggesting that consuming the HP diet from weaning to early adulthood did not protect them against the obesigenic effects of the HFHS diet. In fact, although final body weight did not differ between C and HP rats, the markedly higher body fat in the HP rats is metabolically harmful and suggests that a significant shift in nutrient partitioning occurred in these rats. On the other hand by consuming the high prebiotic diet throughout growth, HF rats were protected against excessive weight gain and fat mass accumulation when challenged with the HFHS diet. And even though the HF rats entered the HFHS diet challenge at a lower body weight they did not catch up this weight during the high energy feeding as weight gain remained significantly lower in HF versus HP rats during the final six weeks of the study. The mechanisms responsible for these contrasting protective and detrimental effects are not completely know but could involve changes in metabolism.
The expression of certain genes involved in glucose and lipid metabolism was altered with diet and these could exert effects that ultimately influence body composition. HMG-CoA reductase is the rate-limiting enzyme in cholesterol synthesis. Serum cholesterol can be reduced by administration of HMG-CoA reductase inhibitors and these are often prescribed to achieve reductions in LDL cholesterol . We observed a significant decrease in HMG-CoA reductase mRNA expression in the liver of rats fed a long-term diet of high fiber which persisted even after both groups consumed the HFHS diet. A HFHS diet has been shown to increase hepatic HMG-CoA reductase mRNA in rats . Given that all of our rats were fed HFHS diet for the last 6 weeks of the study one might anticipate that HMG-CoA reductase would increase in all rats similarly but it is clear that the residual effects of the high fiber diet consumed throughout growth persisted in these animals to minimize this response.
Another potential drug target for lowering cholesterol is cholesterol 7α-hydroxylase . Cholesterol can be incorporated into bile salts by cholesterol 7α-hydroxylase, the initial and rate-determining enzyme for bile synthesis . We demonstrated that a high protein diet, when followed by a diet high in energy results in decreased expression of cholesterol 7α-hydroxylase mRNA which could be indicative of decreased incorporation of cholesterol into bile acids and the potential for longer-term impairment in cholesterol metabolism in these rats.
Glucokinase (GK) catalyzes the rate-limiting step in glycolysis, the phosphorylation of glucose to glucose-6-phospate. In the liver, GK activity determines the rate of glucose utilization and glycogen synthesis . GK first appears in the liver of rats about 16 days after birth and reaches adult activity levels 10-12 days later . There is little research on the effect of long-term high protein diets on glucokinase or GLUT2 activity. We demonstrated an increase in the expression of both genes in the liver due to a long-term weaning diet high in protein subsequently followed by the high energy diet. Although not demonstrated consistently, a high fat diet has been shown to increase hepatic GK mRNA [49, 51]. While some would suggest that increased GK activity could improve hepatic insulin resistance , others have shown that long-term increased GK activity leads to glucose intolerance and hepatic lipid accumulation in mice . Mice over-expressing GK were also more sensitive to the diabetogenic effects of a high fat diet, which supports the elevated triglyceride content seen in our HP rats. Whether or not the increased GK mRNA seen in our high protein fed rats also played a role in the overall exacerbated negative response they had to the HFHS diet warrants further investigation.
The fructose transporter, GLUT5, is expressed in greatest concentration in the small intestine, but is also expressed in the kidneys, skeletal muscle and certain areas of the brain . In rats, GLUT5 expression remains low until the completion of weaning and a switch to solid food . It has been shown in individuals with type 2 diabetes that GLUT5 expression is higher in skeletal muscle  and in the intestines . However, these findings are not consistent and remain controversial . GLUT5 synthesis is regulated very quickly by diet as consumption of fructose results in an increase in the transporter within a few hours . In the intestine of our rats fed a high protein diet we see a general down-regulation in the hexose transporters which likely reflects the lower carbohydrate content of this diet.
When we previously examined the expression of genes controlling glucose and lipid metabolism in young rats (days 7, 14, 21, 28, 35 of age) weaned onto high protein or fiber diets, we observed minimal change in gene expression in the intestine, and in the liver a decrease solely in FAS mRNA with HP and HF compared to control . On the other hand, the greater GLP-1 levels we observed in these same young rats with high fiber diet  did persist into adulthood as demonstrated in the current study. Considered together, these studies comparing early and late changes to the diets would suggest that while some metabolic pathways (satiety hormones) are influenced very early on by diet, others (expression of genes regulating lipid metabolism) are only exasperated by a high energy diet in adulthood. The greater hepatic triglyceride content and fat mass in the high protein rats in this study provide phenotypic evidence for changes occurring at the level of gene expression.