Select human milk oligosaccharide supplementation in post-weanling rats affects metabolism and gut microbiota into adulthood.

OBJECTIVE: Feeding infants with human milk versus formula can produce long-lasting benefits, including reduced risk of inflammatory diseases. Most infant formulas do not contain human milk oligosaccharides (HMOs), which are important carbohydrates in human breast milk displaying prebiotic properties. The study's aim was to examine the effect of select HMOs in the post-weaning period. METHODS: Metabolic and microbial outcomes were measured in female and male rats whose post-weaning diet was supplemented with 3'sialyllactose and 2'-O-fucosyllactose (low dose, 0.75% of each, or high dose, 2% of each). It was determined whether exposure to the HMOs would attenuate metabolic dysfunction associated with a high fat/sucrose (HFS) diet. RESULTS: HMO supplementation resulted in dose-dependent and sex-dependent effects, with the high HMO female group displaying reduced food intake and percentage body fat and an increase in Blautia abundance. Early life HMO supplementation did not prevent the effects of an HFS diet on metabolic outcomes; however, rats that received high dose HMOs followed by the HFS diet were the only HFS group that maintained a measurable abundance of Blautia. CONCLUSIONS: This study highlights the potential for HMOs in follow-up infant formulas. Further investigation should include variable HMO mixtures and doses to optimize infant nutrition at this critical stage.

Dietary fiber combinations to mitigate the metabolic, microbial, and cognitive imbalances resulting from diet-induced obesity in rats.

Dietary fiber promotes a healthy gut microbiome and shows promise in attenuating the unfavorable microbial changes resulting from a high-fat/sucrose (HFS) diet. High-fiber diets consisting of oligofructose alone (HFS/O) or in combination with ß-glucan (HFS/OB), resistant starch (HFS/OR), or ß-glucan and resistant starch (HFS/OBR) were fed to diet-induced obese rats for 8 weeks to determine if these fibers could attenuate the obese phenotype. Only the HFS/O group displayed a decrease in body weight and body fat, but all fiber interventions improved insulin sensitivity and cognitive function. The HFS/O diet was the least effective at improving cognitive function and only the HFS/OB group showed improvements in glucose tolerance, thus highlighting the differential effects of fiber types. Hippocampal cytokines (IL-6, IL-10) were more pronounced in the HFS/OB group which coincided with the most time spend in the open arms of the elevated plus maze. All fiber groups showed an increase in beneficial Bifidobacterium and Lactobacillus abundance while the HFS group showed higher abundance of Clostridium. Fecal microbiota transplant from fiber-treated rats into germ-free mice did not alter body composition in the mice but did result in a higher abundance of Bacteroides in the HFS/O and HFS/OB groups compared to HFS. The HFS/OB recipient mice also had higher insulin sensitivity compared to the other groups. This study highlights the influence of dietary fiber type on metabolic and cognitive outcomes suggesting that the type of supplementation (single or combined fibers) could be tailored to specific targeted outcomes.

Effect of supplementation with select human milk oligosaccharides on artificially reared newborn rats.

Early life nutrition fundamentally influences neonatal development and health. Human milk oligosaccharides (HMO) are key components of breast milk but not standard infant formula that support the establishment of the newborn gut microbiota. Using an artificial rearing system, our objective was to test the effect of two HMO on the whole body and organ growth, adiposity, glucose tolerance and faecal microbiota in young rat pups. From postnatal days 4 to 21, Sprague-Dawley rats were randomised to receive one of: (1) CTR (rat milk substitute); (2) 2'FL (CTR + 1·2 g/l 2'-fucosyllactose); (3) 3'SL (CTR + 1·2 g/l 3'-sialyllactose) and (4) 2'FL + 3'SL (CTR + 0·6 g/l 2'-FL + 0·6 g/l 3'-SL). Body weight (BW), bowel movements and food intake were monitored daily, faecal samples collected each week and oral glucose tolerance, body composition and organ weight measured at weaning. No significant differences were observed between groups in growth performance, body composition, organ weight and abundance of dominant faecal microbes. A decreased relative abundance of genus Proteus in week 1 faecal samples and Terrisporobacter in week 3 faecal samples (P < 0·05) was suggestive of a potential pathogen inhibitory effect of 3'SL. Longitudinal changes in the faecal microbiota of artificially reared suckling rats were primarily governed by age (P = 0·001) and not affected by the presence of 2'-FL and/or 3'-SL in rat milk substitutes (P = 0·479). Considering the known protective effects of HMO, further investigation of supplementation with these and other HMO in models of premature birth, extremely low BW or malnutrition may show more pronounced outcomes.

Paternal high protein diet modulates body composition, insulin sensitivity, epigenetics, and gut microbiota intergenerationally in rats.

Mounting evidence demonstrates that paternal diet programs offspring metabolism. However, the contribution of a pre-conception paternal high protein (HP) diet to offspring metabolism, gut microbiota, and epigenetic changes remains unclear. Here we show that paternal HP intake in Sprague Dawley rats programs protective metabolic outcomes in offspring. Compared to paternal high fat/sucrose (HF/S), HP diet improved body composition and insulin sensitivity and improved circulating satiety hormones and cecal short-chain fatty acids compared to HF/S and control diet (P < .05). Further, using 16S rRNA gene sequencing to assess gut microbial composition, we observed increased alpha diversity, distinct bacterial clustering, and increased abundance of Bifidobacterium, Akkermansia, Bacteroides, and Marvinbryantia in HP fathers and/or male and female adult offspring. At the epigenetic level, DNMT1and 3b expression was altered intergenerationally. Our study identifies paternal HP diet as a modulator of gut microbial composition, epigenetic markers, and metabolic function intergenerationally.

C-section birth increases offspring obesity risk dependent on maternal diet and obesity status in rats.

OBJECTIVE: The gut microbiota is a complex ecosystem that shapes host metabolism, especially in early life. Maternal vaginal and gut microbiota is vertically transmitted to offspring during natural birth. Offspring born by cesarean section (CS) do not receive these bacteria and exhibit higher obesity risk later in life. The objective of this study was to examine differences in obesity risk between offspring born naturally (NB) or by CS to lean/obese dams. METHODS: Lean and obese rat dams gave birth to offspring naturally or by CS. Offspring obesity risk was analyzed via body weight/composition, food intake, sucrose preference, gut microbiota, and gene expression in gut and brain tissues. RESULTS: Obese (O)+CS offspring showed greater weight gain and caloric intake but a reduction in hypothalamic agouti related neuropeptide, neuropeptide Y, and interleukin 1ß expression compared with O+NB offspring. Lean (L)+CS offspring had higher serum corticosterone concentration and reduced liver peroxisome proliferator-activated receptor γ expression compared with L+NB. O+CS offspring had long-term alterations to gut microbiota, including increased relative abundance of Faecalibaculum and reduced Muribaculaceae. CONCLUSIONS: Overall, CS alters obesity risk differentially based on maternal obesity status. Further studies looking at the risks of obesity associated with CS are needed, with special attention paid to maternal obesity status and gut microbiota.

The Effects of Human Milk Oligosaccharide Supplementation During Critical Periods of Development on the Mesolimbic Dopamine System.

Human milk oligosaccharides (HMO)s are a key component in human milk and represent an important dietary modulator of infant gut microbiota composition and associated gut-brain axis development and homeostasis. The brain reward system, specifically the mesolimbic dopamine (DA) projections from the ventral tegmental area (VTA) to nucleus accumbens (NAc) is involved in the motivation and preference for food. The objective of the present study was to determine if HMO fortified diets given during the critical period of reward system development (p21) could affect the structure of the reward system. At weaning (p21), Sprague-Dawley rats were randomized to one of four fortified diet groups: Control, 3'sialyllactose (3'FL), 2'-fucosyllactose (2'FL), or a combination of 3'SL and 2'FL (3'SL + 2'FL). Messenger RNA (mRNA) expression was quantified for DA and appetite associated markers in the VTA and NAc and western blots measured the immediate early gene FosB and its isoform ΔFosB. Females fed the 3'SL + 2'FL fortified diet displayed a decrease in DAT expression in the VTA and an increase in leptin expression in the NAc. Females displayed an overall lower expression of NAc D2, VTA ghrelinR, and VTA leptin. In males, VTA DAT and FosB were negatively correlated with body weight and systemic leptin. Sex differences in the expression of DA markers underscore the need to investigate this phenomenon and understand the functional significance in preventing or treating obesity. This study highlights sex differences in response to HMO supplementation and the need for further investigations into the functional significance of nutritional interventions during DA system development.

Prebiotic Oligofructose Prevents Antibiotic-Induced Obesity Risk and Improves Metabolic and Gut Microbiota Profiles in Rat Dams and Offspring.

SCOPE: Antibiotics in early life disrupt microbiota and increase obesity risk. Dietary agents such as prebiotics may reduce obesity risk. The authors examine how antibiotics administered with/without prebiotic oligofructose, alter metabolic and microbial outcomes in pregnant rats and their offspring. METHODS AND RESULTS: Pregnant rats are randomized to: 1) Control, 2) Antibiotic (ABT), 3) Prebiotic (PRE), 4) Antibiotic+Prebiotic (ABT+PRE) during the 3rd week of pregnancy and lactation. Offspring were fed a high fat/high sucrose (HFS) diet from 9-17 weeks of age to unmask obesity risk. ABT dams had higher body weight, body fat and leptin during lactation than all other groups. Prebiotics attenuate these outcomes and increase cecal Bifidobacterium. ABT offspring have higher body weight, fat mass, and liver triglycerides after HFS diet, with a stronger phenotype in males; prebiotics attenuate these. At weaning, male ABT offspring have lower Lactobacillus while PRE and ABT+PRE offspring had higher Bifidobacterium and Collinsella. Fecal microbiota transfer of adult offspring cecal matter could not reliably transfer the obese ABT phenotype. CONCLUSIONS: Antibiotic use during pregnancy/lactation increases adiposity and impairs post-partum weight loss in dams. Co-administering prebiotics with antibiotics in rat dams prevented obesity risk in offspring and is associated with altered gut microbiota.

Different periods of forced abstinence after instrumental learning for food reward of different macronutrient value on responding for conditioned cues and AMPAr subunit levels.

Food craving can be viewed as an intense desire for a specific food that propagates seeking and consuming behavior. Prolonged forced abstinence from rewarding foods can result in escalated food-seeking behavior as measured via elevated responding for food-paired cues in the absence of the primary reward. Palatable food consumption and food-seeking is associated with changes in the abundance and composition of AMPA receptors in the nucleus accumbens (NAc) but differing results have been reported. The present study examined whether different food types could produce escalated food-seeking behavior after various abstinence periods and whether this was associated with changes in AMPA receptor protein levels. Rats were trained for 10 days to bar press for purified, sucrose, or chocolate-flavored sucrose pellets. Rats were tested at 24 hrs, 7 d or 14 d whereby bar pressing resulted in presentation of cues paired with food but no food reward was delivered. Western blotting was used to determine protein levels of GluR1, GluR1pSer845, and GluR2 in the NAc. Three separate groups were assessed: 1) a group that was trained on the operant task and tested for conditioned responding (tested group); 2) a group that was trained on the operant task but not tested (non-tested group); 3) a group that was neither trained nor tested (control). The purified food group showed a time-dependent elevation in conditioned bar pressing over the 3 abstinence periods. GluR1 AMPAr subunit levels were higher in the purified and sucrose groups tested at 24 hours compared to the non-tested and control values. GluR1 levels subsequently declined at the 7- and 14-day abstinence periods in the purified and sucrose tested and non-tested groups compared to control values. GluR2 and pSer845 Glur1 levels were similar across all groups and abstinence periods. These results show that food-seeking behavior associated with forced abstinence from different food rewards may depend on the macronutrient composition of the food reward or the food type given during the abstinence period. A clear link with AMPAr subunit levels in this model was not established.

Exploring time-dependent changes in conditioned place preference for food reward and associated changes in the nucleus accumbens.

The conditioned place preference (CPP) procedure has been used to study the incubation of craving phenomenon with rewarding drugs such as cocaine and methamphetamine. The present study examined whether rats trained in a CPP behavioral design would display an incubation of craving response for chocolate-flavored pellets or milk chocolate chips at the behavioral and neural levels. Rats were conditioned using an unbiased CPP design then underwent abstinence from food reward for 24 hs, 7, 14, or 28 days at which point they were tested for CPP. Brains underwent immunohistochemical staining for c-Fos and FosB as well as Golgi staining to assess dendritic spine density in the nucleus accumbens (NAc). A time-dependent increase in CPP and entries into the previously paired compartment was observed in the chocolate-flavored pellet group but not the milk chocolate group. Time-dependent neural changes were not directly associated with behavioral outcomes but c-Fos labelling was higher in the chocolate pellet group than controls at the 7-day abstinence period. The behavioral results show that chocolate pellets are rewarding and are associated with long-term behavioral changes but, as evidenced by limited neural changes, these food rewards do not have the same effects on the NAc as drugs of abuse.

Comparison of the Time-Dependent Changes in Immediate Early Gene Labeling and Spine Density Following Abstinence From Contingent or Non-contingent Chocolate Pellet Delivery.

Rationale: Incubation of craving is a phenomenon whereby responding for cues associated with a reward increases over extended periods of abstinence. Both contingent and non-contingent behavioral designs have been used to study the incubation of craving phenomenon with differing results. The present study directly compares behavioral and neural changes following contingent or non-contingent administration of chocolate flavored pellets. Objective: The current study examined whether an incubation of craving response would be observed at the behavioral and neural levels following delays of abstinence from chocolate pellets in a contingent or non-contingent reinforcement design. Methods: Rats were trained for 10 days to bar press for chocolate pellets (contingent) or received chocolate pellets in a non-contingent design (classical conditioning). Groups were then subjected to abstinence from the reward for 24 h, 7, 14 or 28 days at which point they were tested for responding for reward associated cues. Following the test, brains from all rats were processed and assessed for c-Fos and FosB labeling as well as dendritic spine density in the nucleus accumbens (NAc). Results: Behavioral measures during the test (lever presses, food hopper entries and locomotor activity) revealed similar behavioral outcomes across all delays indicating the lack of an incubation of craving response on both the contingent and non-contingent designs. Overall, labeling of c-Fos in the NAc was lower for the non-contingent group compared to the operant-trained and food restricted control. Compared to the operant-trained and non-trained control groups, a significantly reduced FosB labeling was noted in the NAc of the classically conditioned groups across all abstinence periods. Spine density in the NAc was elevated in both the classically and operant conditioned compared to the food-restricted, non-trained controls. Conclusions: Chocolate pellet reward did not result in incubation of craving but did produce behavioral learning that was associated with increased spine density. This suggests that chocolate pellet administration results in long-term structural and functional changes that are present for at least 28 days following abstinence. Contingent and non-contingent administration resulted in differential immediate early gene labeling in the NAc, but the functional significance of this has yet to be elucidated.