AGEs are derived from both exogenous and endogenous sources, and the rate at which AGEs accumulate in the body is dependent to a large extent on the chronological age, lifestyle and metabolic health of an individual. Smoking cigarettes and consuming foods containing high concentrations of AGEs (and their precursors) increases the accumulation of AGEs from exogenous sources. Endogenous AGE formation is accelerated under conditions of hyperglycaemia, dyslipidaemia and increased oxidative stress, conditions that are common in individuals with diabetes and in those at risk of developing type 2 diabetes (such as those with prediabetes and the metabolic syndrome) . Moreover, in individuals with impaired renal function, urinary AGE excretion may be diminished resulting in a greater accumulation of AGEs in the body .
Risk factors for the development of type 2 diabetes include obesity, hypertension and cardiovascular disease; conditions that are commonly associated with unhealthy lifestyles including poor food habits. Restricting the intake of foods high in AGEs might potentially reduce AGE accumulation, but adherence to such diets can be challenging given that foods high in AGEs are very palatable due to their enhanced flavour, colour and aroma .
Supplementation of the diet with bifidogenic prebiotic fibres (such as inulin) may reduce or retard the accumulation of AGEs in individuals at risk of developing type 2 diabetes. Prebiotics have been shown to improve and restore optimal microbial balance within the gastrointestinal tract, potentially reducing AGE absorption and/or production by the human host. Preliminary investigations indicate that consuming a high-AGE diet is sufficient to favour the proliferation of potentially pathogenic colonic bacteria over more beneficial species. Consumption of glycated proteins , fried meats  and toasted wheat flakes  encouraged the preferential growth of greater numbers of detrimental gram negative and sulphate-reducing colonic micro-organisms when compared to control diets. Short Chain Fatty Acids (SCFAs) produced as a bacterial by-product of prebiotic fermentation act to lower the intestinal pH, inhibiting the growth of protein-degrading micro-organisms capable of producing potentially toxic metabolites. SCFAs also stimulate colonic smooth muscle contractions, speeding intestinal transit and limiting the time available for protein fermentation and putrefaction to occur in the gut . Therapeutic manipulation of the gut microbiota with prebiotics may restore gut normobiosis and reduce AGE accumulation in humans at risk for type 2 diabetes development by the following mechanisms:
Maintenance of gut barrier function
Kinetic studies have estimated that up to 30% of dietary AGEs consumed are intestinally absorbed . Under circumstances of increased intestinal permeability, it is likely that greater quantities of dietary AGEs and their reactive dicarbonyl precursors may be able to gain entry into the systemic circulation. Elevated levels of circulating proinflammatory cytokines and ROS frequently observed in individuals with prediabetes are known to compromise tight junctions between cells, disrupting the integrity of the intestinal barrier and enabling the absorption of larger, potentially deleterious compounds . Prebiotic fermentation products may reduce gastrointestinal permeability and as a result limit the absorption of exogenous AGEs.
The SCFAs acetate and butyrate are intricately involved in the maintenance of gut epithelial integrity. Acetate increases colonic blood flow and butyrate is the principal fuel for colonocytes, assisting to optimise epithelial cell health . Butyrate reduces gastrointestinal permeability by enhancing the activation of the peroxisomal proliferator-activated receptor gamma (PPARgamma) gene, a nuclear receptor involved in the attenuation of inflammation in colonic epithelial cells . Butyrate also upregulates the expression of mucin-associated genes important in maintaining the integrity of the intestinal mucosal barrier . Oligofructose supplementation in mice has been shown to increase the expression of zonula and occludin, proteins important in the maintenance of tight junctions between gastrointestinal cells , and a dietary inulin intervention reduced markers of intestinal permeability in healthy adult males . Butyrate is a histone deacetylase inhibitor and is likely to promote the transcription of these tight junction proteins. Prebiotic-induced changes in gut microbiota also increase endogenous production of Glucagon-like peptide 2 (GLP-2), which enhances gut barrier function by promoting the proliferation of crypt cells [71, 72].
Reduction of oxidative stress, inflammation and insulin resistance
Increased production of ROS stimulate endogenous AGE formation by oxidising glucose and unsaturated fatty acids to generate reactive dicarbonyls. Experimental drug treatments which attenuate oxidative stress have previously demonstrated reductions in serum AGE levels . Cytokine production at sites of inflammation stimulate immune cell activation of NADPH oxidase (NOX) and production of myeloperoxidase, enzymes involved in the oxidation of amino acids to form AGE precursors . Activated immune cells can also secrete the high-mobility group box 1 (HMGB-1) protein which is capable of binding to RAGE, thereby inducing further inflammation .
A high fat diet (independent of the level of obesity in the host) is associated with negative changes in bacterial communities within the colon . In response to a high fat meal, bacterial lipopolysaccharide (LPS) translocates from the intestine into the host circulation, resulting in ‘metabolic endotoxemia’ [77, 78]. LPS is a major component of the outer membrane of gram negative bacteria, and is a potent activator of the mammalian immune system. LPS interaction with immune cells stimulates macrophage over-production of ROS, enhances the secretion of pro-inflammatory cytokines, and contributes to weight gain and development of insulin resistance [79, 80]. Individuals with type 2 diabetes have been found to possess endotoxemia levels 2-fold higher than people without diabetes . Metabolic endotoxemia also positively correlates with total energy intake and fasting insulin levels in the general population . In mice with high fat diet-induced metabolic endotoxemia, nutritional supplementation of the diet with prebiotics restored intestinal levels of gram positive bacteria (particularly Bifidobacterium species) and subsequently improved glucose tolerance and reduced circulating concentrations of LPS and pro-inflammatory cytokines . Human trials involving dietary prebiotic supplementation have successfully reduced serum LPS levels  and markers of lipid peroxidation (a process which generates AGE precursors), possibly through the reduction of ROS production or the direct antioxidant ability of some Bifidobacterium and Lactobacillus bacterial species [84, 85].
SCFAs produced as a bacterial by-product of prebiotic fermentation are absorbed into the host circulation, effecting the expression of a wide range of genes in distal tissues associated with cell proliferation, differentiation and apoptosis. SCFAs are ligands for the G-protein coupled receptors (GPRs) GPR41 and GPR43  on immune cells. These receptors are involved in down-regulating inappropriate immune cell production of pro-inflammatory cytokines, chemokines and ROS . The SCFAs acetate, proprionate and butyrate exhibit a variety of anti-inflammatory actions through inhibition of NFκB activation, prevention of LPS-stimulated TNFα production in neutrophils and suppression of cytokine production [31, 88–90].
Numerous other immune modulating effects have been observed secondary to gut bacterial activity including the production of anti-inflammatory compounds such as polysaccharide A , peptidoglycan  and conjugated linoleic acid , and the induction of T-regulatory cells .
Promotion of weight reduction
An energy-restricted diet resulting in weight loss has been shown to reduce serum AGE levels in overweight and obese individuals . The consumption of prebiotics in human clinical trials has promoted self-reported satiety , weight reduction, reduced production of the orexigenic hormone grehlin and stimulated expression of the appetite-reducing hormone peptide YY (PYY) . Interaction with GPR41 by the SCFAs proprionate and butyrate increases satiety , upregulates PYY production and modulates the expression of leptin, a hormone important in controlling energy intake and expenditure .
In mice, the selective growth of certain lactobacillus species in the colon reduced body fat storage through the up-regulation of fiaf (fasting induced adipose factor) gene expression and inhibition of lipoprotein lipase (LPL) [98–100]. These findings may have the potential to affect weight reduction in humans and subsequently reduce circulating AGE levels.
Enhanced antioxidant capacity
Inulin enhances the proliferation of lactic acid producing bacteria capable of synthesising B-group vitamins, some of which have an antioxidant capacity . These vitamins can be utilised by the human host to neutralise ROS. Vitamins B1 and B6 trap the carbonyl groups of highly reactive AGE precursors before they can react with proteins . Some Lactobacillus and Bifidobacterium species are efficient scavengers of the lipid peroxidation product malandialdehyde, protecting the host from excessive accumulation of this toxic AGE precursor . Inulin also exhibits antioxidant properties independent of altering gut bacterial growth and is able to scavenge a number of ROS, which may help to reduce lipid peroxidation in the stomach .
The SCFA butyrate, produced as a bacterial by-product of inulin fermentation, has been shown to increase colonic glutathione production . Glutathione is an antioxidant co-factor required for glyoxalase I activity, an enzyme which degrades the AGE precursor methylglyoxyl. Increased production of ROS is also thought to deplete glutathione levels. Through the reduction of oxidative stress, prebiotics may assist in the maintainance or upregulation of the glyoxylase pathway.
Reduction of hyperglycemia
Both transient and chronic elevations in blood glucose increase endogenous AGE generation. Activation of GPR43 in adipocytes by proprionate inhibits lipolysis and lowers glycemia in healthy individuals . Butyrate has been shown to reverse diet-induced insulin resistance in animal studies , possibly by enhancing PPARgamma expression which increases fatty acid oxidation in muscle.
Glucagon-like peptide 1 (GLP-1) is an incretin hormone released from intestinal L-cells in response to consumption of carbohydrates and fats. GLP-1 potentiates glucose-induced insulin secretion, reducing post-prandial blood glucose levels. GLP-1 also enhances satiety and slows gastric emptying. Prebiotic feeding in rats promoted L-cell differentiation in the colon and increased GLP-1 production [107, 108], probably through an increase in bacterial production of butyrate.
The consumption of inulin as a dietary supplement may also influence circulating AGE concentrations independently of its prebiotic function. High glycemic index (GI) diets and their resultant hyperglycemic effect have been shown to enhance AGE formation in healthy individuals . Inulin is a soluble fibre which when consumed daily may play a role in the reduction of the GI of the diet. Many soluble fibres are known for their ability to delay gastric emptying and slow the rate of intestinal nutrient absorption, reducing the GI of the carbohydrates in the meal. Regular consumption of prebiotic soluble fibre reduces fasting and post-prandial serum glucose levels in people with impaired glucose tolerance  and type 2 diabetes .
● To our knowledge, this is the first trial to investigate the potential effects of gut bacterial modulation on advanced glycation.
● Random allocation of participants to treatment sequence and intention to treat analysis will ensure study bias is minimised.
● Double-blind crossover placebo-controlled trial.
● Dietary data will be obtained to determine dietary patterns as well as estimates of total energy intake, fat and AGE consumption.
● CML in this study will be measured using a validated ELISA method.
● Dietary AGE consumption will be estimated from an open-source food AGE database, containing AGE values obtained using validated measurement techniques.
Type 2 diabetes is a chronic condition which often develops over decades, making it difficult to conduct intervention studies using the presence or absence of diabetes as the primary biological end-point. This study will measure surrogate biochemical markers of early type 2 diabetes pathogenesis, which could be considered a limitation of the trial. Long-term studies will need to be conducted in order to confirm the results of this research.
This study will measure serum CML as an indicator of AGE concentration in blood samples. Multiple other forms of AGEs exist, many of which have not yet been characterised, so the findings of this trial cannot be applied to all members of the AGE family. However, serum CML concentration shows a moderate to high correlation with other known circulating AGEs .
Applicability of research findings
In Australia, conservative estimates predict that at least 2 million adults will have been diagnosed with type 2 diabetes by 2025 . The burden of disease associated with diabetes has a substantial impact on costs associated directly with health care as well as loss of productivity and decreased quality of life. The widespread consumption of high fat, heat processed foods and the increasing prevalence of obesity in Australia warrant simple interventions including those that prevent AGE-mediated damage. If dietary treatments aimed at altering the gut microbiota prove to be effective strategies for preventing or slowing the development of type 2 diabetes, they could become mainstream therapies for individuals with diabetes risk factors.