The impact of whey protein consumption and exercise on the composition and diversity of the gut microbiota: a high through-put DNA sequencing approach

Advances in culture independent technologies over the last decade have highlighted the pivotal role which the gut microbiota plays in maintaining human health. Conversely, perturbations to the composition or actions of the ‘normal/functioning’ microbiota have been frequently associated with the pathogenesis of several disease states. Therefore the selective modulation of enteric microbial communities represents a viable target for the development of novel treatments for such diseases. Notably, while bovine whey proteins and exercise have been shown to positively influence several physiological processes, such as energy balance, their effect on the composition or functionality of the gut microbiota remains largely unknown. In this thesis, a variety of ex vivo, murine and human models are used in conjunction with high-throughput DNA sequencing-based analysis to provide valuable and novel insights into the impact of both whey proteins and exercise on enteric microbial communities. Overall the results presented in this thesis highlight that the consumption both whey protein isolate (WPI), and individual component proteins of whey such as bovine serum albumin (BSA) and lactoferrin, reduce high fat diet associated body weight gain and are associated with beneficial alterations within the murine gut microbiota. Although the impact of exercise on enteric microbial communities remains less clear, it may be that longer term investigations are required for the true effect of exercise on the gut microbiota to be fully elucidated.

A study of diet and health in the elderly; the gut microbiota as a source of bioactive agents

The global proportion of older persons is increasing rapidly. Diet and the intestinal microbiota independently and jointly contribute to health in the elderly. The habitual dietary patterns and functional microbiota components of elderly subjects were investigated in order to identify specific effector mechanisms. A study of the dietary intake of Irish community-dwelling elderly subjects showed that the consumption of foods high in fat and/or sugar was excessive, while consumption of dairy foods was inadequate. Elderly females typically had a more nutrient- dense diet than males and a considerable proportion of subjects, particularly males, had inadequate intakes of calcium, magnesium, vitamin D, folate, zinc and vitamin C. The association between dietary patterns, glycaemic index and cognitive function was also investigated. Elderly subjects consuming ‘prudent’ dietary patterns had better cognitive function compared to those consuming ‘Western’ dietary patterns. Furthermore, fully-adjusted regression models revealed that a high glycaemic diet was associated with poor cognitive function, demonstrating a new link between nutrition and cognition. An extensive screening study of the elderly faecal-derived microbiota was also undertaken to examine the prevalence of antimicrobial production by intestinal bacteria. A number of previously characterised bacteriocins were isolated (gassericin T, ABP-118, mutacin II, enterocin L-50 and enterocin P) in this study. Interestingly, a Lactobacillus crispatus strain was found to produce a potentially novel antimicrobial compound. Full genome sequencing of this strain revealed the presence of three loci which exhibited varying degrees of homology with the genes responsible for helveticin J production in Lb. helveticus. An additional study comparing the immunomodulatory capacity of ‘viable’ and ‘non-viable’ Bifidobacterium strains found that Bifidobacterium-fermented milks (BFMs) containing ‘non-viable’ cells could stimulate levels of IL-10 and TNF-α in a manner similar to those stimulated by BFMs containing ‘viable’ cells in vitro.

Postprandial serum endotoxin in healthy humans is modulated by dietary fat in a randomized, controlled, cross-over study

Background: High-fat diets may contribute to metabolic disease via postprandial changes in serum endotoxin and inflammation. It is unclear how dietary fat composition may alter these parameters. We hypothesized that a meal rich in n-3 (ω3) fatty acids would reduce endotoxemia and associated inflammation but a saturated or n-6 (ω6) fatty acid-rich meal would increase postprandial serum endotoxin concentrations and systemic inflammation in healthy adults. Methods: Healthy adults (n = 20; mean age 25 ± 3.2 S.D. years) were enrolled in this single-blind, randomized, cross-over study. Participants were randomized to treatment and reported to the laboratory, after an overnight fast, on four occasions separated by at least one week. Participants were blinded to treatment meal and consumed one of four isoenergetic meals that provided: 1) 20 % fat (control; olive oil) or 35 % fat provided from 2) n-3 (ω3) (DHA = 500 mg; fish oil); 3) n-6 (ω6) (7.4 g; grapeseed oil) or 4) saturated fat (16 g; coconut oil). Baseline and postprandial blood samples were collected. Primary outcome was defined as the effect of treatment meal on postprandial endotoxemia. Serum was analyzed for metabolites, inflammatory markers, and endotoxin. Data from all 20 participants were analyzed using repeated-measures ANCOVA. Results: Participant serum endotoxin concentration was increased during the postprandial period after the consumption of the saturated fat meal but decreased after the n-3 meal (p < 0.05). The n-6 meal did not effect a different outcome in participant postprandial serum endotoxin concentration from that of the control meal (p > 0.05). There was no treatment meal effect on participant postprandial serum biomarkers of inflammation. Postprandial serum triacylglycerols were significantly elevated following the n-6 meal compared to the n-3 meal. Non-esterified fatty acids were significantly increased after consumption of the saturated fat meal compared to other treatment meals. Conclusions: Meal fatty acid composition modulates postprandial serum endotoxin concentration in healthy adults. However, postprandial endotoxin was not associated with systemic inflammation in vivo. Trial registration: This study was retrospectively registered at clinicaltrials.gov as NCT02521779 on July 28, 2015.

The impact of a variety of factors on the obesity associated gut microbiota

The obesity pandemic has become perhaps the most prevalent health issue of our time, with more than 10% of the world’s population now being obese. Obesity can be defined as abnormal or excess fat accumulation that may impair health and results from an imbalance between energy intake and energy expenditure. A decrease in physical activity due to an increase in sedentary forms of work, changing modes of transport and increasing urbanization is likely a major contributory factor. Diet is another major factor with the increased availability and intake of calorie dense, high fat foods being of global concern. Notably, with respect to this thesis, over the last decade advances in the field of next generation sequencing (NGS) have facilitated investigations to determine the relationship between the gut microbiota and obesity. This thesis examines the impact of a variety of factors on the obesity associated gut microbiota. Overall the results presented in this thesis highlight that microbial diversity is influenced by diet, exercise, antibiotics and disease state, however it is only through further understanding of the structure and function that we can identify targets that can impact on health.

Obesity, diet quality and absenteeism in a working population

The relationship between workplace absenteeism and adverse lifestyle factors (smoking, physical inactivity and poor dietary patterns) remains ambiguous. Reliance on self-reported absenteeism and obesity measures may contribute to this uncertainty. Using objective absenteeism and health status measures, the present study aimed to investigate what health status outcomes and lifestyle factors influence workplace absenteeism. Cross-sectional data were obtained from a complex workplace dietary intervention trial, the Food Choice at Work Study. Four multinational manufacturing workplaces in Cork, Republic of Ireland. Participants included 540 randomly selected employees from the four workplaces. Annual count absenteeism data were collected. Physical assessments included objective health status measures (BMI, midway waist circumference and blood pressure). FFQ measured diet quality from which DASH (Dietary Approaches to Stop Hypertension) scores were constructed. A zero-inflated negative binomial (zinb) regression model examined associations between health status outcomes, lifestyle characteristics and absenteeism. The mean number of absences was 2·5 (sd 4·5) d. After controlling for sociodemographic and lifestyle characteristics, the zinb model indicated that absenteeism was positively associated with central obesity, increasing expected absence rate by 72 %. Consuming a high-quality diet and engaging in moderate levels of physical activity were negatively associated with absenteeism and reduced expected frequency by 50 % and 36 %, respectively. Being in a managerial/supervisory position also reduced expected frequency by 50 %. To reduce absenteeism, workplace health promotion policies should incorporate recommendations designed to prevent and manage excess weight, improve diet quality and increase physical activity levels of employees.

The role played by angiotensin (1-7) in the regulation of renal haemodynamics and excretory function in anesthetized rats

Initial studies have demonstrated that intra- renal infusion of Ang (1-7) caused a diuresis and natriuresis that was proportional to the degree of activation of the Renin Angiotensin Aldosterone System (RAAS). This raised the question as why the magnitude of this diuresis and natriuresis was compromised in rats receiving a high sodium diet (suppressed RAAS) and enhanced in low sodium fed rats (activated RAAS)? Could the answer lie with changes in intra-renal AT1 or Mas receptor expression? Interestingly, the observed Ang (1-7) induced increases in sodium and water excretion in rats receiving either a low or normal sodium diet were and blocked in the presence of the AT 1 receptor antagonist (Losartan) in the presence of the, 'Mas' receptor antagonist (A-779). These data suggest that both AT1 and 'Mas' receptors need to be functional in order to fully mediate the renal responses to intra-renal Ang (1-7) infusion. Importantly, further experimentation also revealed that there is a proportional relationship between AT 1 receptor expression in the rat renal cortex and the magnitude of the excretory actions of intra renal Ang (1-7) infusion, which is only partially dependent on the level of 'Mas' receptor expression. These observations suggest that although Ang (1-7) induced increases in sodium and water excretion are mediated by the Mas receptor, the magnitude of these excretory responses appear to be dependent upon the level of AT 1 receptor expression and more specifically Ang II/ AT 1 receptor signalling. Thus in rats receiving a low sodium diet, Ang (1-7) acts via the Mas receptor to inhibit Ang II/ AT 1 receptor signalling. In rats receiving a high sodium diet the down regulated AT 1 receptor expression implies a reduction in Ang II/ AT 1 receptor signalling which renders the counter-regulatory effects of intra-renal Ang (1-7) infusion redundant.

The impact of whey protein isolate on energy balance regulation

Using C57BL/6J mice fed whey protein isolate (WPI) enriched high fat (HFD) or low-fat diets (LFD), this study tested the hypothesis that WPI directly impacts on adiposity by influencing lipid metabolism. WPI suppressed HFD-induced body fat and increased lean mass at 8 weeks of dietary challenge despite elevated plasma triacylglycerol (TAG) levels, suggesting reduced TAG storage. WPI reduced HFD-associated hypothalamic leptin and insulin receptor (IR) mRNA expression, and prevented HFD-associated reductions in adipose tissue IR and glucose transporter 4 expression. These effects were largely absent at 21 weeks of HFD feeding, however WPI increased lean mass and cause a trend towards decreased fat mass, with notable increased Lactobacillus and decreased Clostridium gut bacterial species. Increasing the protein to carbohydrate ratio enhanced the above effects, and shifted the gut microbiota composition away from the HFD group. Seven weeks of WPI intake with a LFD decreased insulin signalling gene expression in the adipose tissue in association with an increased fat accumulation. WPI reduced intestinal weight and length, suggesting a potential functional relationship between WPI, gastro-intestinal morphology and insulin related signalling in the adipose. Extending the study to 15 weeks, did not affect adipose fat weight, but decreased energy intake, weight gain and intestinal length. The functionality of protein sensing lysophosphatidic acid receptor 5 (LPA5) in 3T3-L1 pre-adipocytes was assessed. Over-expression of the receptor in 3T3-L1 pre-adipocytes provided a growth advantage to the cells and suppressed cellular differentiation into mature fat cells. In conclusion, the data demonstrates WPI impacts on adiposity by influencing lipid metabolism in a temporal manner, resulting possibly due to changes in lean mass, hypothalamic and adipose gene expression, gut microbiota and gastrointestinal morphology. The data also showed LPA5 is a novel candidate in regulating of preadipocyte growth and differentiation, and may mediate dietary protein effects on adipose tissue.

Longitudinal study of changes in body mass index, anthropometric measures, dietary intake and physical activity in cohorts of school-going Irish adolescents

The prevalence of obesity worldwide has increased dramatically over the last few decades. Poor dietary habits and low levels of exercise in adolescence are often maintained into adulthood where they can impact on the incidence of obesity and chronic diseases. A 3-year longitudinal study of anthropometric, dietary and exercise parameters was carried out annually (2005 - 2007) in 3 Irish secondary schools. Anthropometric measurements were taken in each year and analysed longitudinally. Overweight and obesity were at relatively low levels in these adolescents. Height, weight, BMI, waist and hip circumferences and TST increased significantly over the 3 years. Waist-to-hip ratio (WHR) decreased significantly over time. Boys were significantly taller than girls across the 3 years. A 3-day weighed food diary was used to assess food intake by the adolescents. Analysis of dietary intake data was determined using WISP©. Mean daily energy and nutrient intakes were reported. Mean daily energy and macronutrient intakes were analysed longitudinally. The adolescents’ diet was characterised by relatively high saturated fat intakes and insufficient fruit and vegetable consumption. The dietary pattern did not change significantly over the 3 years. Boys consumed more energy than girls over the study period. A validated questionnaire was used to assess physical activity and sedentary activity levels. Boys were substantially more active and had higher energy expenditure estimates than girls throughout the study. A significant longitudinal decrease in physical activity levels among the adolescents was observed. Both genders spent more than the recommended amount of time (hrs/day) pursing sedentary activities. The dietary pattern in these Irish adolescents is relatively poor. Of additional concern is the overall longitudinal decrease in physical activity levels. Promoting consumption of a balanced diet and increased exercise levels among adolescents will help to reduce future public health care costs due to weight-related diseases.