Gut Microbiota Composition and Predicted Microbial Metabolic Pathways of Obesity Prone and Obesity Resistant Outbred Sprague-Dawley CD Rats May Account for Differences in Their Phenotype.

Like humans, outbred Sprague-Dawley CD rats exhibit a polygenic pattern of inheritance of the obese phenotype and not all individuals exposed to a high calorie intake develop obesity. We hypothesized that differences in gut microbiota composition account for phenotype differences between obese prone (OP) and obese resistant (OR) rats. We studied the gut microbiota composition of OPand OR rats after a high fat (HF) diet and how they respond to fermentation of resistant starch (RS). In phase 1 of the study 28 OP and 28 OR rats were fed a HF diet. In order to determine causal role of microbiota on phenotypes, In phase 2, a microbiota transplant between the two phenotypes was performed before switching all rats to a HF diet supplemented with 20% RS. We determined microbiota composition by 16S sequencing and predicted microbiota function by PICRUSt2. Despite a similar calorie intake, in phase 2 OP rats gained more weight and accumulated more abdominal fat in both phase 1 and 2 compared to OR rats (P < 0.001; n = 6). The OP rats fermented RS more robustly compared with OR rats with an increase in total bacteria, short chain fatty acids, and increased weight of the cecum, but microbiota of OP rats had much lower alpha diversity and evenness. The microbiota of OP rats, had higher amounts of bacteria from order Bacteroidales, specifically family Muribaculaceae (S24-7), which is known to possess several starch degrading enzymes and was reported in previous studies to increase with fermentation of RS. The OR rats fermented RS less but had higher bacterial diversity and evenness and had significantly higher bacterial counts from phylum Firmicutes particularly order Clostridiales, genus Clostridium and an uncultured bacterium of the genus Akkermansia. The microbiota of OR rats had enhanced bacterial chemotaxis, phosphotransferase system (PTS), and fatty acid biosynthesis compared to OP rats whose microbiota had higher glycan degradation and LPS biosynthesis pathways. The microbiota transplant did not change obesity phenotype or microbiota composition. In conclusion, a higher alpha-diversity and evenness of the microbiota and higher proportions of Clostridiales and Akkermansia in OR rats were associated with a better metabolic phenotype with lower body fat. However, robust RS fermentation caused a lower diversity and evenness and did not result in a leaner phenotype.

Differences in Capacity of High-Amylose Resistant Starch, Whole-Grain Flour, and a Combination of Both to Modify Intestinal Responses of Male Sprague Dawley Rats Fed Moderate and High Fat Diets.

Gastrointestinal tract (GIT) responses to a high-amylose resistant starch (RS) product were compared to those observed when RS was combined with whole grain (WG) and to controls with low RS intake in rats fed moderate or high fat diets. Regardless of fat intake, rats fed RS or WG + RS diets had higher cecum weights, higher intestinal quantities of short chain fatty acids, and lower intestinal content pH, and their GIT cells had increased gene expression for gluconeogenesis and barrier function compared to controls. Whereas RS resulted in greater GIT content acetate and propionate and lowest pH, the WG + RS diets yielded higher butyrate. Rats fed the RS diet with MF had higher cecum weights than those fed either the RS diet with HF or the WG + RS diet with either MF or HF. Diets containing combinations of RS and other dietary fibers should be considered for RS-mediated GIT benefits.

Abundance of the species Clostridium butyricum in the gut microbiota contributes to differences in obesity phenotype in outbred Sprague-Dawley CD rats.

OBJECTIVES: Gut microbiota profiles contribute to differences in obesity phenotype. We examined the abundance of the species Clostridium butyricum in relation to obesity phenotype. METHODS: In outbred Sprague -Dawley rats we examined effects of dietary fat, resistant starch (RS), and a microbiota transplant on obesity phenotype. Using targeted qPCR, we examined the abundance of total gut bacteria and C. butyricum in relation to the propensity of obesity prone and obesity resistant rats to accumulate abdominal fat. RESULTS: Before inclusion of dietary RS, obesity resistant (OR) rats had higher amounts of total bacteria, and C. butyricum compared to obesity prone (OP) rats (P < 0.005 in study I, P < 0.0001 in study II). A high fat diet (HF) lowered C. butyricum levels while RS had no effect. Dietary RS elicited robust fermentation and increased total bacteria only in OP rats. In preparation for the transplant, antibiotics were administered to recipient rats. Four weeks thereafter, total bacteria levels were restored but, C. butyricum levels were not. The transplant between the two phenotypes had no effect on abundance of C. butyricum and obesity phenotype. CONCLUSIONS: While C. butyricum is a known saccharolytic, its proliferation is not enhanced by fermentation of resistant starch. C. butyricum maybe one of the species that constitute a core microbiota involved in energy storage and metabolism through mechanisms that are not yet known.

Novel Resistant Starch Type 4 Products of Different Starch Origins, Production Methods, and Amounts Are Not Equally Fermented when Fed to Sprague-Dawley Rats.

SCOPE: The possible mechanisms of production of four novel resistant starch type 4 (RS4) products for total cecal fermentation in an in vivo rodent model are evaluated. METHODS AND RESULTS: Forty weanling rats are randomly assigned to five groups (n = 8) for a 3-week study. Starches are the RS type 4 products, as 10% of weight of RS diets (RSA-RSD), and AMIOCA starch (100% amylopectin) comprises 53.6% weight of control (CON) and 43.6% weight of RS diets. The RS products vary by percent purity and origin (potato, corn, tapioca). At euthanasia, cecal contents, serum, GI tract, and abdominal fat are collected. RSB, RSC, and RSD fed rats have greater empty cecum weights, lower cecal content pH, higher cecal content wet weight, and higher total cecal content acetate and propionate than the CON and RSA fed rats. Two other indicators of fermentation, total cecal contents butyrate and glucagon-like peptide 1, do not have significant ANOVA F values, which require more subjects for 80% power. CONCLUSION: RS4 products that are produced from different starch origins with varying amounts of RS4 content and different methods of production are not uniformly fermented in an in vivo model.

CD Obesity-Prone Rats, but not Obesity-Resistant Rats, Robustly Ferment Resistant Starch Without Increased Weight or Fat Accretion.

OBJECTIVE: This study used CD obesity-prone (OP) and obesity-resistant (OR) rats to examine how weight gain and fat accretion relate to fermentation levels and microbiota composition after feeding resistant starch (RS). METHODS: After feeding OP rats and OR rats a high-fat (HF) diet for 4 weeks, rats were stratified into three groups: they were fed either an HF diet (group 1: HF-HF) or were switched to a low-fat (LF) diet (group 2: HF-LF) or an LF diet supplemented with 20% RS by weight for 4 weeks (group 3: HF-LFRS). Energy intake, body weight, fermentation variables, and microbiota composition were determined. RESULTS: In OP rats, RS elicited robust fermentation (increased cecal contents, short-chain fatty acids, and serum glucagon-like peptide 1). Total bacteria, species of the Bacteroidales family S24-7, and the archaean Methanobrevibacter smithii increased. The robust fermentation did not elicit higher weight or fat accretion when compared with that of control rats fed the same isocaloric diets (HF-LF ± RS). In OR rats, body weight and fat accretion were also not different between HF-LF ± RS diets, but RS elicited minimal changes in fermentation and microbiota composition. CONCLUSIONS: Robust fermentation did not contribute to greater weight. Fermentation levels and changes in microbiota composition in response to dietary RS differed by obesity phenotype.

Simultaneous delivery of antibiotics neomycin and ampicillin in drinking water inhibits fermentation of resistant starch in rats.

SCOPE: Antibiotics ampicillin 1 g/L and neomycin 0.5 g/L were added to drinking water before or during feeding of resistant starch (RS) to rats to inhibit fermentation. METHODS AND RESULTS: In a preliminary study, antibiotics and no RS were given prior to rats receiving a transplant of cecal contents via gavage from donor rats fed RS (without antibiotics) or a water gavage before feeding resistant starch to both groups. Antibiotics given prior to feeding RS did not prevent later fermentation of RS regardless of either type of gavage. In the second study, antibiotics were given simultaneously with feeding of RS. This resulted in inhibition of fermentation of RS with cecal contents pH >8 and low amounts of acetate and butyrate. Rats treated with antibiotics had reduced Bifidobacteria spp., but similar Bacteroides spp. to control groups to reduce acetate and butyrate and preserve the production of propionate. Despite reduced fermentation, rats given antibiotics had increased glucagon-like peptide 1 (GLP-1) and cecum size, measures that are usually associated with fermentation. CONCLUSIONS: A simultaneous delivery of antibiotics inhibited fermentation of RS. However, increased GLP-1 and cecum size would be confounding effects in assessing the mechanism for beneficial effects of dietary RS by knocking out fermentation.

Obese ZDF rats fermented resistant starch with effects on gut microbiota but no reduction in abdominal fat.

SCOPE: To determine if whole-grain (WG) flour with resistant starch (RS) will produce greater fermentation than isolated RS in obese Zucker Diabetic Fatty (ZDF) rats, and whether greater fermentation results in different microbiota, reduced abdominal fat, and increased insulin sensitivity. METHODS AND RESULTS: This study utilized four groups fed diets made with either isolated digestible control starch, WG control flour (6.9% RS), isolated RS-rich corn starch (25% RS), or WG corn flour (25% RS). ZDF rats fermented RS and RS-rich WG flour to greatest extent among groups. High-RS groups had increased serum glucagon-like peptide 1 (GLP-1) active. Feeding isolated RS showed greater Bacteroidetes to Firmicutes phyla among groups, and rats consuming low RS diets possessed more bacteria in Lactobacillus genus. However, no differences in abdominal fat were observed, but rats with isolated RS had greatest insulin sensitivity among groups. CONCLUSIONS: Data demonstrated ZDF rats (i) possess a microbiota that fermented RS, and (ii) WG high-RS fermented better than purified RS. However, fermentation and microbiota changes did not translate into reduced abdominal fat. The defective leptin receptor may limit ZDF rats from responding to increased GLP-1 and different microbiota for reducing abdominal fat, but did not prevent improved insulin sensitivity.

Biodistribution and toxicity of orally administered poly (lactic-co-glycolic) acid nanoparticles to F344 rats for 21 days.

AIM: Quantify the biodistribution and assess the toxicity of PLGA (poly-lactic-co-glycolic acid) and surface-modified PLGA chitosan (PLGA/Chi) nanoparticles (NPs) orally administered for 7, 14 and 21 days to F344 rats. MATERIALS & METHODS: Fluorescent NPs were tracked in F344 rat tissues, and toxicity was evaluated by alkaline phosphatase and alanine transaminase levels, and by histologic examination of tissue samples. RESULTS: Biodistribution of PLGA and PLGA/Chi were similar, with highest amounts found in the intestine and liver. Alkaline phosphatase increased significantly in treated rats. Mild histological differences were detected in the intestine and liver. CONCLUSION: PLGA and PLGA/Chi NPs behaved similarly presenting minimal toxicity in the liver and intestine, but not in kidney, lung and brain.

Role of resistant starch in improving gut health, adiposity, and insulin resistance.

The realization that low-glycemic index diets were formulated using resistant starch led to more than a decade of research on the health effects of resistant starch. Determination of the metabolizable energy of the resistant starch product allowed for the performance of isocaloric studies. Fermentation of resistant starch in rodent studies results in what appears to be a healthier gut, demonstrated by increased amounts of short-chain fatty acids, an apparent positive change in the microbiota, and increased gene expression for gene products involved in normal healthy proliferation and apoptosis of potential cancer cells. Additionally, consumption of resistant starch was associated with reduced abdominal fat and improved insulin sensitivity. Increased serum glucagon-like peptide 1 (GLP-1) likely plays a role in promoting these health benefits. One rodent study that did not use isocaloric diets demonstrated that the use of resistant starch at 8% of the weight of the diet reduced body fat. This appears to be approximately equivalent to the human fiber requirement. In human subjects, insulin sensitivity is increased with the feeding of resistant starch. However, only 1 of several studies reports an increase in serum GLP-1 associated with resistant starch added to the diet. This means that other mechanisms, such as increased intestinal gluconeogenesis or increased adiponectin, may be involved in the promotion of improved insulin sensitivity. Future research may confirm that there will be improved health if human individuals consume the requirement for dietary fiber and a large amount of the fiber is fermentable.

Photoactivated miR-148b-nanoparticle conjugates improve closure of critical size mouse calvarial defects.

Inducible systems providing temporal control of differentiation have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. The aim of this study was to demonstrate that nanoformulated microRNA (miRNA) conjugates activated via photo exposure can lead to the induced osteogenic differentiation of human adipose-derived stromal/stem cells (hASCs) in vivo. The conjugate PC-miR-148b-SNP, a mimic of miRNA-148b tethered to silver nanoparticles (SNPs) via a photolabile linker, was used to modulate gene expression for improved closure of a critical size defect drilled on the right parietal bone of male CD-1 nude homozygous mice. The PC-miR-148b-SNP conjugates added to hASCs and loaded to either Matrigel or polycaprolactone (PCL) scaffolds resulted in different levels of healing of the defect. After 4 and 12weeks, 3-D micro-computed tomography reconstructed images indicate statistically significant defect closure from 3.83±1.19% to 5.46±2.01% and 6.54±4.28% to 32.53±8.3% for non-photoactivated and photoactivated conjugates, respectively, in the PCL scaffolds. The results were confirmed with H&E and Masson's Trichrome stains in the transverse sections of photoactivated conjugates. Collagen fiber staining was greatest at 12weeks when it reached approximately the same density and thickness as the native calvarium. This technology provides a platform that can be used with other miRNAs that actively govern the pathways responsible for regenerative and wound healing processes.

Biodistribution of PLGA and PLGA/chitosan nanoparticles after repeat-dose oral delivery in F344 rats for 7 days.

AIM: To quantify in vivo the biodistribution of poly(lactic-co-glycolic) acid (PLGA) and PLGA/chitosan nanoparticles (PLGA/Chi NPs) and assess if the positive charge of chitosan significantly enhances nanoparticle absorption in the GI tract. MATERIAL & METHODS: PLGA and PLGA/Chi NPs covalently linked to tetramethylrhodamine-5-isothiocyanate (TRITC) were orally administered to F344 rats for 7 days, and the biodistribution of fluorescent NPs was analyzed in different organs. RESULTS: The highest amount of particles (% total dose/g) was detected for both treatments in the spleen, followed by intestine and kidney, and then by liver, lung, heart and brain, with no significant difference between PLGA and PLGA/Chi NPs.  CONCLUSION: Only a small percentage of orally delivered NPs was detected in the analyzed organs. The positive charge conferred by chitosan was not sufficient to improve the absorption of the PLGA/Chi NPs over that of PLGA NPs.