Nondigestible Fructans Alter Gastrointestinal Barrier Function, Gene Expression, Histomorphology, and the Microbiota Profiles of Diet-Induced Obese C57BL/6J Mice.

BACKGROUND: Obesity is associated with compromised intestinal barrier function and shifts in gastrointestinal microbiota that may contribute to inflammation. Fiber provides benefits, but impacts of fiber type are not understood. OBJECTIVE: We aimed to determine the impact of cellulose compared with fructans on the fecal microbiota and gastrointestinal physiology in obese mice. METHODS: Eighteen-wk-old male diet-induced obese C57BL/6J mice (n = 6/group; 40.5 g) were fed high-fat diets (45% kcal fat) containing 5% cellulose (control), 10% cellulose, 10% short-chain fructooligosaccharides (scFOS), or 10% inulin for 4 wk. Cecal and colon tissues were collected to assess barrier function, histomorphology, and gene expression. Fecal DNA extracts were subjected to 16S ribosomal RNA amplicon-based Illumina MiSeq sequencing to assess microbiota. RESULTS: Body weight gain was greater (P < 0.05) in scFOS-fed than in 10% cellulose-fed mice. Both groups of fructan-fed mice had greater (P < 0.05) cecal crypt depth (scFOS: 141 µm; inulin: 145 µm) than both groups of cellulose-fed mice (5% and 10%: 109 µm). Inulin-fed mice had greater (P < 0.05) cecal transmural resistance (101 Ω × cm(2)) than 5% cellulose-fed controls (45 Ω × cm(2)). Inulin-fed mice had lower (P < 0.05) colonic mRNA abundance of Ocln (0.41) and Mct1 (0.35) than those fed 10% cellulose (Ocln: 1.28; Mct1: 0.90). Fructan and cellulose groups had different UniFrac distances of fecal microbiota (P < 0.05) and α diversity, which demonstrated lower (P < 0.01) species richness in fructan-fed mice. Mice fed scFOS had greater (P < 0.05) Actinobacteria (15.9%) and Verrucomicrobia (Akkermansia) (17.0%) than 5% controls (Actinobacteria: 0.07%; Akkermansia: 0.08%). Relative abundance of Akkermansia was positively correlated (r = 0.56, P < 0.01) with cecal crypt depth. CONCLUSIONS: Fructans markedly shifted gut microbiota and improved intestinal physiology in obese mice, but the mechanisms by which they affect gut integrity and inflammation in the obese are still unknown.

Genome-wide association mapping combined with reverse genetics identifies new effectors of low water potential-induced proline accumulation in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) exhibits natural genetic variation in drought response, including varying levels of proline (Pro) accumulation under low water potential. As Pro accumulation is potentially important for stress tolerance and cellular redox control, we conducted a genome-wide association (GWAS) study of low water potential-induced Pro accumulation using a panel of natural accessions and publicly available single-nucleotide polymorphism (SNP) data sets. Candidate genomic regions were prioritized for subsequent study using metrics considering both the strength and spatial clustering of the association signal. These analyses found many candidate regions likely containing gene(s) influencing Pro accumulation. Reverse genetic analysis of several candidates identified new Pro effector genes, including thioredoxins and several genes encoding Universal Stress Protein A domain proteins. These new Pro effector genes further link Pro accumulation to cellular redox and energy status. Additional new Pro effector genes found include the mitochondrial protease LON1, ribosomal protein RPL24A, protein phosphatase 2A subunit A3, a MADS box protein, and a nucleoside triphosphate hydrolase. Several of these new Pro effector genes were from regions with multiple SNPs, each having moderate association with Pro accumulation. This pattern supports the use of summary approaches that incorporate clusters of SNP associations in addition to consideration of individual SNP probability values. Further GWAS-guided reverse genetics promises to find additional effectors of Pro accumulation. The combination of GWAS and reverse genetics to efficiently identify new effector genes may be especially applicable for traits difficult to analyze by other genetic screening methods.

Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation.

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ(1)-pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation.

Intrinsic aerobic capacity impacts susceptibility to acute high-fat diet-induced hepatic steatosis.

Aerobic capacity/fitness significantly impacts susceptibility for fatty liver and diabetes, but the mechanisms remain unknown. Herein, we utilized rats selectively bred for high (HCR) and low (LCR) intrinsic aerobic capacity to examine the mechanisms by which aerobic capacity impacts metabolic vulnerability for fatty liver following a 3-day high-fat diet (HFD). Indirect calorimetry assessment of energy metabolism combined with radiolabeled dietary food was employed to examine systemic metabolism in combination with ex vivo measurements of hepatic lipid oxidation. The LCR, but not HCR, displayed increased hepatic lipid accumulation in response to the HFD despite both groups increasing energy intake. However, LCR rats had a greater increase in energy intake and demonstrated greater daily weight gain and percent body fat due to HFD compared with HCR. Additionally, total energy expenditure was higher in the larger LCR. However, controlling for the difference in body weight, the LCR has lower resting energy expenditure compared with HCR. Importantly, respiratory quotient was significantly higher during the HFD in the LCR compared with HCR, suggesting reduced whole body lipid utilization in the LCR. This was confirmed by the observed lower whole body dietary fatty acid oxidation in LCR compared with HCR. Furthermore, LCR liver homogenate and isolated mitochondria showed lower complete fatty acid oxidation compared with HCR. We conclude that rats bred for low intrinsic aerobic capacity show greater susceptibility for dietary-induced hepatic steatosis, which is associated with a lower energy expenditure and reduced whole body and hepatic mitochondrial lipid oxidation.

Comparison of Diet versus Exercise on Metabolic Function and Gut Microbiota in Obese Rats.

UNLABELLED: Cardiometabolic impairments that begin early in life are particularly critical, because they often predict metabolic dysfunction in adulthood. Obesity, high-fat diet (HFD), and inactivity are all associated with adipose tissue (AT) inflammation and insulin resistance (IR), major predictors of metabolic dysfunction. Recent evidence has also associated the gut microbiome with cardiometabolic health. PURPOSE: The objective of this study is to compare equal energy deficits induced by exercise and caloric reduction on cardiometabolic disease risk parameters including AT inflammation, IR, and gut microbiota changes during HFD consumption. METHODS: Obesity-prone rats fed HFD were exercise trained (Ex, n = 10) or weight matched to Ex via caloric reduction although kept sedentary (WM, n = 10), and compared with ad libitum HFD-fed (Sed, n = 10) rats for IR, systemic energetics and spontaneous physical activity (SPA), adiposity, and fasting metabolic parameters. Visceral, subcutaneous, periaortic, and brown AT (BAT), liver, aorta, and cecal digesta were examined. RESULTS: Despite identical reductions in adiposity, Ex, but not WM, improved IR, increased SPA by approximately 26% (P < 0.05 compared with WM and Sed), and reduced LDL cholesterol (P < 0.05 compared with Sed). WM and Ex both reduced inflammatory markers in all AT depots and aorta, whereas only Ex increased indicators of mitochondrial function in BAT. Ex significantly increased the relative abundance of cecal Streptococcaceae and decreased S24-7 and one undefined genus in Rikenellaceae; WM induced similar changes but did not reach statistical significance. CONCLUSIONS: Both Ex and WM reduced AT inflammation across depots, whereas Ex caused more robust changes to gut microbial communities, improved IR, increased fat oxidation, increased SPA, and increased indices of BAT mitochondrial function. Our findings add to the growing body of literature indicating that there are weight-loss-independent metabolic benefits of exercise.

Efficient Targeting of Adipose Tissue Macrophages in Obesity with Polysaccharide Nanocarriers.

Obesity leads to an increased risk for type 2 diabetes, heart disease, stroke, and cancer. The causal link between obesity and these pathologies has recently been identified as chronic low-grade systemic inflammation initiated by pro-inflammatory macrophages in visceral adipose tissue. Current medications based on small-molecule drugs yield significant off-target side effects with long-term use, and therefore there is a major need for targeted therapies. Here we report that nanoscale polysaccharides based on biocompatible glucose polymers can efficiently target adipose macrophages in obese mice. We synthesized a series of dextran conjugates with tunable size linked to contrast agents for positron emission tomography, fluorophores for optical microscopy, and anti-inflammatory drugs for therapeutic modulation of macrophage phenotype. We observed that larger conjugates efficiently distribute to visceral adipose tissue and selectively associate with macrophages after regional peritoneal administration. Up to 63% of the injected dose remained in visceral adipose tissue 24 h after administration, resulting in >2-fold higher local concentration compared to liver, the dominant site of uptake for most nanomedicines. Furthermore, a single-dose treatment of anti-inflammatory conjugates significantly reduced pro-inflammatory markers in adipose tissue of obese mice. Importantly, all components of these therapeutic agents are approved for clinical use. This work provides a promising nanomaterials-based delivery strategy to inhibit critical factors leading to obesity comorbidities and demonstrates a unique transport mechanism for drug delivery to visceral tissues. This approach may be further applied for high-efficiency targeting of other inflammatory diseases of visceral organs.

High-Fat Diet Alters Serum Fatty Acid Profiles in Obesity Prone Rats: Implications for In Vitro Studies.

High-fat diets (HFD) are commonly used in rodents to induce obesity, increase serum fatty acids and induce lipotoxicity in various organs. In vitro studies commonly utilize individual free fatty acids (FFA) to study lipid exposure in an effort to model what is occurring in vivo; however, these approaches are not physiological as tissues are exposed to multiple fatty acids in vivo. Here we characterize circulating lipids in obesity-prone rats fed an HFD in both fasted and fed states with the goal of developing physiologically relevant fatty acid mixtures for subsequent in vitro studies. Rats were fed an HFD (60% kcal fat) or a control diet (10% kcal fat) for 3 weeks; liver tissue and both portal and systemic blood were collected. Fatty acid profiles and absolute concentrations of triglycerides (TAG) and FFA in the serum and TAG, diacylglycerol (DAG) and phospholipids in the liver were measured. Surprisingly, both systemic and portal serum TAG were ~40% lower in HFD-fed compared to controls. Overall, compared to the control diet, HFD feeding consistently induced an increase in the proportion of circulating polyunsaturated fatty acids (PUFA) with a concomitant decline in monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) in both serum TAG and FFA. The elevations of PUFA were mostly attributed to increases in n-6 PUFA, linoleic acid and arachidonic acid. In conclusion, fatty acid mixtures enriched with linoleic and arachidonic acid in addition to SFA and MUFA should be utilized for in vitro studies attempting to model lipid exposures that occur during in vivo HFD conditions.

Physical Activity Differentially Affects the Cecal Microbiota of Ovariectomized Female Rats Selectively Bred for High and Low Aerobic Capacity.

The gut microbiota is considered a relevant factor in obesity and associated metabolic diseases, for which postmenopausal women are particularly at risk. Increasing physical activity has been recognized as an efficacious approach to prevent or treat obesity, yet the impact of physical activity on the microbiota remains under-investigated. We examined the impacts of voluntary exercise on host metabolism and gut microbiota in ovariectomized (OVX) high capacity (HCR) and low capacity running (LCR) rats. HCR and LCR rats (age = 27 wk) were OVX and fed a high-fat diet (45% kcal fat) ad libitum and housed in cages equipped with (exercise, EX) or without (sedentary, SED) running wheels for 11 wk (n = 7-8/group). We hypothesized that increased physical activity would hinder weight gain, increase metabolic health and shift the microbiota of LCR rats, resulting in populations more similar to that of HCR rats. Animals were compared for characteristic metabolic parameters including body composition, lipid profile and energy expenditure; whereas cecal digesta were collected for DNA extraction. 16S rRNA gene-based amplicon Illumina MiSeq sequencing was performed, followed by analysis using QIIME 1.8.0 to assess cecal microbiota. Voluntary exercise decreased body and fat mass, and normalized fasting NEFA concentrations of LCR rats, despite only running one-third the distance of HCR rats. Exercise, however, increased food intake, weight gain and fat mass of HCR rats. Exercise clustered the gut microbial community of LCR rats, which separated them from the other groups. Assessments of specific taxa revealed significant (p<0.05) line by exercise interactions including shifts in the abundances of Firmicutes, Proteobacteria, and Cyanobacteria. Relative abundance of Christensenellaceae family was higher (p = 0.026) in HCR than LCR rats, and positively correlated (p<0.05) with food intake, body weight and running distance. These findings demonstrate that exercise differentially impacts host metabolism and gut microbial communities of female HCR and LCR rats without ovarian function.

Differential effects of low-fat and high-fat diets on fed-state hepatic triacylglycerol secretion, hepatic fatty acid profiles, and DGAT-1 protein expression in obese-prone Sprague-Dawley rats.

The purpose of this study was to compare the effects of short-term low-fat (LF) and high-fat (HF) diets on fed-state hepatic triacylglycerol (TAG) secretion, the content of proteins involved in TAG assembly and secretion, fatty acid oxidation (FAO), and the fatty acid profile of stored TAG. Using selectively bred obese-prone Sprague-Dawley rats, we directly measured fed-state hepatic TAG secretion, using Tyloxapol (a lipoprotein lipase inhibitor) and a standardized oral mixed meal (45% carbohydrate, 40% fat, 15% protein) bolus in animals fed a HF or LF diet for 2 weeks, after which the rats were maintained on their respective diet for 1 week (washout) prior to the liver being excised to measure protein content, FAO, and TAG fatty acid profiles. Hepatic DGAT-1 protein expression was ∼27% lower in HF- than in LF-fed animals (p < 0.05); the protein expression of all other molecules was similar in the 2 diets. The fed-state hepatic TAG secretion rate was ∼39% lower (p < 0.05) in HF- (4.62 ± 0.18 mmol·h(-1)) than in LF- (7.60 ± 0.57 mmol·h(-1)) fed animals. Hepatic TAG content was ∼2-fold higher (p < 0.05) in HF- (1.07 ± 0.15 nmol·g(-1) tissue) than in LF- (0.50 ± 0.16 nmol·g(-1) tissue) fed animals. In addition, the fatty acid profile of liver TAG in HF-fed animals closely resembled the diet, whereas in LF-fed animals, the fatty acid profile consisted of mostly de novo synthesized fatty acids. FAO was not altered by diet. LF and HF diets differentially alter fed-state hepatic TAG secretion, hepatic fatty acid profiles, and DGAT-1 protein expression.

A comparison of inflammatory and oxidative stress markers in adipose tissue from weight-matched obese male and female mice.

Expansion of intra-abdominal adipose tissue and the accompanying inflammatory response has been put forward as a unifying link between obesity and the development of chronic diseases. However, an apparent sexual dimorphism exists between obesity and chronic disease risk due to differences in the distribution and abundance of adipose tissue. A range of experimental protocols have been employed to demonstrate the role of estrogen in regulating health benefits; however, most studies are confounded by significant differences in body weight and adiposity. Therefore, the purpose of this study was to compare weight-matched obese male and female mice to determine if the sex-dependent health benefits remain when body weight is similar. The development of obesity in female mice receiving a high-fat diet was delayed; however, subsequent comparisons of weight-matched obese mice revealed greater adiposity in obese female mice. Despite excess adiposity and enlarged adipocyte size, obese females remained more glucose tolerant than weight-matched male mice, and this benefit was associated with increased expression of adiponectin and reductions in immune cell infiltration and oxidative stress in adipose tissue. Therefore, the protective benefits of estrogen persist in the obese state and appear to improve the metabolic phenotype of adipose tissue and the individual.