Role of D-fagomine and omega-3 plyunsaturated fatty acids on gut microbia and related metabolic changes in healthy rats and in a model of fat-induced prediabetes

[eng] Modern lifestyles in an increasing number of human societies include consuming an excess of saturated fats and refined sugars as well as enjoying microbiologically aseptic environments. Maintaining the appropriate distribution of gut microbial populations (eubiosis) is emerging as an attractive approach to prevent ectopic fat accumulation and insulin resistance (IR), and to avert the progression of diabetes. Functional food components are those that help maintain the normal bodily functions beyond providing energy or building blocks. They may bring about physiological health benefits for both healthy subjects following a healthy lifestyle and people belonging to risk groups such as obese or pre-diabetic populations. D-Fagomine is an iminosugar originally present in buckwheat with the capacity for selectively inhibiting bacterial adhesion to the intestinal mucosa. ω-3 Polyunsaturated fatty acids (PUFAs) reduce blood pressure as well as levels of insulin, triacylglycerols, cholesterol and total lipids. Iminosugars and ω-3 PUFAs may be used as functional food ingredients or dietary supplements to maintain a healthy status over time and to reduce risk factors for diabetes. This thesis focuses on the effects of buckwheat D-fagomine, fish ω-3 PUFAs (EPA/DHA 1:1) and their combination on the gut microbiota and related metabolic variables in the host both in healthy rats and a rat model of fat-induced pre-diabetes. In the first part of this thesis we explored the effects of D-fagomine and ω-3 PUFAs (EPA/DHA 1:1) on rats given a standard diet as a model for healthy subjects. We found that D-fagomine had the capacity for promoting microbial functional diversity by increasing the Bacteroidetes/Firmicutes ratio and for mitigating the age-related reduction in populations of the putatively beneficial Lactobacilliales and Bifidobacteriales. Also, the populations of the genus Prevotella remained stable over time in animals supplemented with D- fagomine, independently of ω-3 PUFAs supplementation. The combination between D-fagomine and ω-3 PUFAs provided the functional benefits of each supplement. Notably, it helped stabilize populations of Prevotella in the rat intestinal tract while reducing weight gain and providing the anti-inflammatory and cardiovascular benefits of ω-3 PUFAs. In the second part we explored the effects of the same supplements on rats with fat-induced pre-diabetes as a model for people at risk of suffering from diabetes and cardiovascular diseases. We found that D-fagomine delayed the development of a fat-induced pre-diabetic state by reducing low-grade inflammation. We suggest that the anti-inflammatory effect of D-fagomine may be linked to a reduction in fat-induced overpopulation of minor gut bacterial groups such as Enterobacteriales. The combined supplements counteracted the high-fat induced incipient IR, and liver inflammation, while increasing the cecal content, the Bacteroidetes/Firmicutes ratio as well as the populations of putatively beneficial Bifidobacteriales and Lactobacilliales. The functional effects of the combination between D-fagomine and EPA/DHA 1:1 against the gut dysbiosis and the very early metabolic alterations induced by a high-fat westernized diet were mainly those of D-fagomine complemented by the anti-inflammatory action of ω-3 PUFAs. The results of this thesis point clearly towards a functional role for D-fagomine in the maintenance of the intestinal health by preserving diversity and mitigating the age-related reduction of some beneficial bacteria and also in the prevention of risk factors for diet-induced pre-diabetes reinforced by the action of ω-3 PUFAs by complementary mechanisms

Eubiotic effect of buckwheat D-fagomine in healthy rats

Diversity and balance of gut microorganisms is fundamental for health throughout life. The aim of this study is to explore the possible eubiotic effect of the buckwheat iminosugar D-fagomine (0.096% w/w in standard feed) in growing healthy Wistar Kyoto rats. Feed and energy intake, residual energy in feces, and body weight gain were independent of D-fagomine supplementation throughout the intervention (24 weeks). The populations of significant bacterial subgroups and species were determined in fecal and cecal DNA by quantitative real-time PCR. D-Fagomine increased the Bacteroidetes:Firmicutes ratio and partially counteracted the loss of Lactobacilliales and Bifidobacteriales over time. The supplementation reduced the levels of excreted short- chain fatty acids (SCFAs) as determined by gas chromatography. This paper provides preliminary evidence that D-fagomine has the capacity to promote microbial functional diversity by increasing the Bacteroidetes:Firmicutes ratio and to mitigate the age-related reduction in populations of the putatively beneficial Lactobacilliales and Bifidobacteriales

Fate of d‑fagomine after oral administration to rats

D-Fagomine is an iminosugar found in buckwheat that is capable of inhibiting the adhesion of potentially pathogenic bacteria to epithelial mucosa and of reducing postprandial blood glucose concentration. This paper evaluates the excretion and metabolism of orally administered D-fagomine in rats and compares outcomes with the fate of 1-deoxynojirimycin. D-Fagomine and 1- deoxynojirimycin show similar absorption and excretion kinetics. D-Fagomine is partly absorbed (41-84%, dose 2 mg/kg body weight) and excreted in urine within 8 h while non-absorbed fraction is cleared in feces within 24 h. D-Fagomine is partially methylated (about 10% in urine and 3% in feces). The concentration of D-fagomine in urine from 1 to 6 h after administration is higher than 10 mg/L, the concentration that inhibits adhesion of Escherichia coli. Orally administered D-fagomine is partially absorbed and then rapidly excreted in urine were it reaches a concentration that may be protective against urinary tract infections

Fiber-like Action of D-Fagomine on the Gut Microbiota and Body Weight of Healthy Rats

The goal of this work is to explore if the changes induced by D-fagomine in the gut microbiota are compatible with its effect on body weight and inflammation markers in rats. Methods: Sprague Dawley rats were fed a standard diet supplemented with D-fagomine (or not, for comparison) for 6 months. The variables measured were body weight, plasma mediators of inflammation (hydroxyeicosatetraenoic acids, leukotriene B4, and IL-6), and the concentration of acetic acid in feces and plasma. The composition and diversities of microbiota in cecal content and feces were estimated using 16S rRNA metabarcoding and high-throughput sequencing. We found that after just 6 weeks of intake D-fagomine significantly reduced body weight gain, increased the plasma acetate concentration, and reduced the plasma concentration of the pro-inflammatory biomarkers' leukotriene B4, interleukin 6 and 12 hydroxyeicosatetraenoic acids. These changes were associated with a significantly increased prevalence of Bacteroides and Prevotella feces and increased Bacteroides, Prevotella, Clostridium, and Dysgonomonas while reducing Anaerofilum, Blautia, and Oribacterium in cecal content. In conclusion, D-fagomine induced changes in the composition and diversity of gut microbiota similar to those elicited by dietary fiber and compatible with its anti-inflammatory and body-weight-reducing effects

Combined buckwheat d-fagomine and fish omega-3 pufas stabilize the populations of gut prevotella and bacteroides while reducing weight gain in rats

Some functional food components may help maintain homeostasis by promoting balanced gut microbiota. Here, we explore the possible complementary effects of d-fagomine and ω-3 polyunsaturated fatty acids (ω-3 PUFAs) eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA 1:1) on putatively beneficial gut bacterial strains. Male Sprague-Dawley rats were supplemented with d-fagomine, ω-3 PUFAs, or both, for 23 weeks. Bacterial subgroups were evaluated in fecal DNA by quantitative real-time polymerase chain reaction (qRT-PCR) and short-chain fatty acids were determined by gas chromatography. We found that the populations of the genus Prevotella remained stable over time in animals supplemented with d-fagomine, independently of ω-3 PUFA supplementation. Animals in these groups gained less weight than controls and rats given only ω-3 PUFAs. d-Fagomine supplementation together with ω-3 PUFAs maintained the relative populations of Bacteroides. ω-3 PUFAs alone or combined with d-fagomine reduced the amount of acetic acid and total short-chain fatty acids in feces. The plasma levels of pro-inflammatory arachidonic acid derived metabolites, triglycerides and cholesterol were lower in both groups supplemented with ω-3 PUFAs. The d-fagomine and ω-3 PUFAs combination provided the functional benefits of each supplement. Notably, it helped stabilize populations of Prevotella in the rat intestinal tract while reducing weight gain and providing the anti-inflammatory and cardiovascular benefits of ω-3 PUFA

Functional effects of the buckwheat iminosugar D-fagomine on rats with diet-induced prediabetes

Scope: The goals of this work were to test if D-fagomine, an iminosugar that reduces body weight gain, can delay the appearance of a fat-induced prediabetic state in a rat model and to explore possible mechanisms behind its functional action. Methods and results: Wistar Kyoto rats were fed a high-fat diet supplemented with D7 fagomine (or not; for comparison) or a standard diet (controls) for 24 weeks. The variables measured were: fasting blood glucose and insulin levels; glucose tolerance; diacylglycerols as intracellular mediators of insulin resistance in adipose tissue, liver and muscle; inflammation markers (plasma IL-6 and leptin, and liver and adipose tissue histology markers); eicosanoids from arachidonic acid as lipid mediators of inflammation; and the populations of Bacteroidetes, Firmicutes, Enterobacteriales and Bifidobacteriales in feces. We found that D-fagomine reduces fat-induced impaired glucose tolerance, inflammation markers and mediators (hepatic microgranulomas and lobular inflammation, plasma IL-6, prostaglandin E2 and leukotriene B4) while attenuating the changes in the populations of Enterobacteriales and Bifidobacteriales. Conclusion: D-Fagomine delays the development of a fat-induced prediabetic state in rats by reducing low-grade inflammation. We suggest that the anti-inflammatory effect of D-fagomine may be linked to a reduction in fat-induced overpopulation of minor gut bacteria

Effects of combined d-fagomine and omega-3 PUFAs on gut microbiota subpopulations and diabetes risk factors in rats fed a high-fat diet

Food contains bioactive compounds that may prevent changes in gut microbiota associated with Westernized diets. The aim of this study is to explore the possible additive effects of d-fagomine and ω-3 PUFAs (EPA/DHA 1:1) on gut microbiota and related risk factors during early stages in the development of fat-induced pre-diabetes. Male Sprague Dawley (SD) rats were fed a standard diet, or a high-fat (HF) diet supplemented with d-fagomine, EPA/DHA 1:1, a combination of both, or neither, for 24 weeks. The variables measured were fasting glucose and glucose tolerance, plasma insulin, liver inflammation, fecal/cecal gut bacterial subgroups and short-chain fatty acids (SCFAs). The animals supplemented with d-fagomine alone and in combination with ω-3 PUFAs accumulated less fat than those in the non-supplemented HF group and those given only ω-3 PUFAs. The combined supplements attenuated the high-fat-induced incipient insulin resistance (IR), and liver inflammation, while increasing the cecal content, the Bacteroidetes:Firmicutes ratio and the populations of Bifidobacteriales. The functional effects of the combination of d-fagomine and EPA/DHA 1:1 against gut dysbiosis and the very early metabolic alterations induced by a high-fat diet are mainly those of d-fagomine complemented by the anti-inflammatory action of ω-3 PUFAs

The buckwheat iminosugar d-fagomine attenuates sucrose-induced steatosis and hypertension in rats

Scope: This study examines the long-term functional effects of D-fagomine on sucrose4 induced factors of metabolic dysfunctions and explores possible molecular mechanisms behind its action. Methods & results: Wistar Kyoto (WKY) rats were fed a 35% sucrose solution with D- fagomine (or not, for comparison) or mineral water (controls) for 24 weeks. We recorded: body weight; energy intake; glucose tolerance; plasma leptin concentration and lipid profile; populations of Bacteroidetes, Firmicutes, bacteroidales, clostridiales, enterobacteriales, and Escherichia coli in feces; blood pressure; urine uric acid and F2t isoprostanes (F2-IsoPs); perigonadal fat deposition; and hepatic histology and diacylglycerols (DAGs) in liver and adipose tissue. D-Fagomine reduced sucrose-induced hypertension, urine uric acid and F2-IsoPs (markers of oxidative stress; OS), steatosis and liver DAGs, without significantly affecting perigonadal fat deposition and impaired glucose tolerance. It also promoted excretion of enterobacteriales generated by the dietary intervention. Conclusion: D-fagomine counteracts sucrose-induced steatosis and hypertension, presumably by reducing the postprandial levels of fructose in the liver

Mechanistically different effects of fat and sugar on insulin resistance, hypertension and gut microbiota in rats

Insulin resistance (IR) and impaired glucose tolerance (IGT) are the first manifestations of diet-induced metabolic alterations leading to type-2 diabetes, while hypertension is the deadliest risk factor of cardiovascular disease. The roles of dietary fat and fructose in the development of IR, IGT and hypertension are controversial. We tested the long-term effects of an excess of fat or sucrose (fructose/glucose) on healthy male Wistar Kyoto (WKY) rats. Fat affects IR and IGT earlier than fructose through low-grade systemic inflammation evidenced by liver inflammatory infiltration, increased levels of plasma interleukin-6, prostaglandin E2 and reduced levels of protective short-chain fatty acids without triggering hypertension. Increased populations of gut Enterobacteriales and Escherichia coli may contribute to systemic inflammation through the generation of lipopolysaccharides. Unlike fat, fructose induces increased levels of diacylglycerols (lipid mediators of IR) in the liver, urine F2-isoprostanes (markers of systemic oxidative stress) and uric acid, and triggers hypertension. Elevated populations of Enterobacteriales and E. coli were only detected in rats given an excess of fructose at the end of the study. Dietary fat and fructose trigger IR and IGT in clearly differentiated ways in WKY rats: early low-grade inflammation and late direct lipid toxicity, respectively; gut microbiota plays a role mainly in fat-induced IR; and hypertension is independent of inflammation55 mediated IR. The results provide evidence which suggests that the combination of fat and sugar is potentially more harmful than fat or sugar alone when taken in excess

Implication of gut microbiota in the physiology of rats intermittently exposed to cold and hypobaric hypoxia

This study examines the influence of intermittent exposure to cold, hypobaric hypoxia, and their combination, in gut microbiota and their metabolites in vivo, and explores their effects on the physiology of the host. Sprague-Dawley rats were exposed to cold (4ºC), hypobaric hypoxia (462 torr), or both simultaneously, 4 h/day for 21 days. Biometrical and hematological parameters were monitored. Gut bacterial subgroups were evaluated by qRT-PCR and short-chain fatty acids were determined by gas chromatography in caecum and feces. Cold increased brown adipose tissue, Clostridiales subpopulation and the concentration of butyric and isovaleric acids in caecum. Hypobaric hypoxia increased hemoglobin, red and white cell counts and Enterobacteriales, and reduced body and adipose tissues weights and Lactobacilliales. Cold plus hypobaric hypoxia counteracted the hypoxia-induced weight loss as well as the increase in white blood cells, while reducing the Bacteroidetes:Firmicutes ratio and normalizing the populations of Enterobacteriales and Lactobacilliales. In conclusion, intermittent cold and hypobaric hypoxia exposures by themselves modified some of the main physiological variables in vivo , while their combination kept the rats nearer to their basal status. The reduction of the Bacteroidetes:Firmicutes ratio and balanced populations of Enterobacteriales and Lactobacilliales in the gut may contribute to this effect