THE IMPACT OF HIGH AND LOW FODMAP CONDITIONS ON BLOOD GLUCOSE CONCENTRATIONS IN HEALTHY YOUNG ADULTS

Objective: Fermentable oligosaccharides, disaccharides, monos accharides and polyols (FODMAPs) are a group of carbohydrates that evade d ig stion in the small intestine and are subsequently metabolized by colonic microbi ota. Colonic fermentation of FODMAPs has been linked to a prebiotic effect resul ting in improved blood glucose response and subjective appetite. The purpose of t his study was to examine how changes in FODMAP consumption effects blood glucose re ponse and subjective appetite. Fasting breath hydrogen was also examine d as an indicator of colonic fermentation. Design: This study utilized a single blind, randomized, cro ss ver design. Healthy participants (n=16) were instructed to follow a low -FODMAP and high-FODMAP diet for a period of three days separated by an 11day “washout period.” Fasting and post-prandial blood glucose were assessed via Chole stech. Subjective appetite was analyzed through use of visual analogue scales. Dat a were analyzed via repeated measures analysis of variance. Results: Blood glucose concentrations did not vary significa ntly between the two dietary interventions (p=.111, η =.155). Reduced total area under the curve (TAUC)

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To be submitted to the Journal of the Academy of Nutrition and Dietetics was seen after the high-FODMAP intervention, however, this decrease was not significant (6722±861 vs. 7149±1120 mg/dl*min, p=.178). No significant changes in subjective appetite were noted. Fasting breath hydrogen did not vary significantly between the two interventions, however, was found to be inversely correlated with glycemic response (r=-0.54, p=.034). Outcomes were reanalyzed utilizing only subjects consuming greater than 4 grams of FODMAPs during high-FODMAP intervention. Reduced post-prandial blood glucose response (F( 1,7df )=7.21,p.007). and reduced blood glucose TAUC was also seen (t=3.60, p=.009) in this subset.

Conclusion:
The High-FODMAP intervention resulted in a non-significant reduction in blood glucose. However, poor dietary compliance likely explains this result. The inverse relationship seen between breath hydrogen and blood glucose TAUC indicate a potential prebiotic effect. Individuals compliant with high FODMAP diet did show reduced glycemic response. Further research, with larger samples and longer interventions, is needed.

Introduction:
Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) are a class of short-chained carbohydrates that are prevalent in the modern diet 1,2 . Common dietary FODMAPs include oligosaccharides such as fructans (or fructooligosacchrides) and galactans (or galactooligosaccharides), disaccharides such as lactose, monomers such as fructose, and polyols such as sorbitol and mannitol 3 . FODMAPs are associated with common functional properties including: limited absorption in the small intestine, high osmotic activity, and rapid fermentation by colonic microbiota [4][5][6] . Due to these functional properties, excess consumption of dietary FODMAPs has been linked to increased incidence of diarrhea, flatulence, as well as abdominal pain and distension amongst individuals with irritable bowel syndrome (IBS) 2,7 . Diets decreasing FODMAP intake have shown the ability to mitigate gastrointestinal symptoms and improve quality of life for individuals with IBS 8,9 .
While reducing dietary FODMAPs has proven to be an effective means to manage IBS, long-term use of a low-FODMAP diet could have adverse health consequences and may not be appropriate for people without IBS. Low-FODMAP foods are generally considered to be high glycemic index (HGI) consisting of simple carbohydrates that are easily digested and absorbed 10 . Elevated intake of HGI foods have been found to increase risk of metabolic disease, diabetes, and cardiovascular disease 10 . Conversely, high-FODMAP foods are associated with a low glycemic index (LGI) 10 . These carbohydrates are not as readily absorbed by the small intestine.
Thus LGI foods reduce postprandial glycemic response, which is important regarding development of chronic diseases such as diabetes and heart disease 11 .
In addition to reduced post-prandial glycemic response, LGI foods have demonstrated the ability to reduce glycemic response at subsequent meals 12,13 . This 'second meal effect' is likely due to a prebiotic effect attributed to increased colonic fermentation [14][15][16] . Malabsorbed carbohydrates, upon entering the colon, are rapidly metabolized, via fermentation reaction, by the resident colonic microbiota. Short-chain fatty acids (SCFA), byproducts of anaerobic metabolism, interact with colonic epithelial cells and have been linked to increased post-prandial expression of numerous gut peptide hormones, most notably glucagon like peptide -1 (GLP-1), which has been shown to have an important role regarding the improvement of glycemic response 17,18 . Increased colonic fermentation, as measured by breath hydrogen, is inversely related to blood glucose response 19,20 . Furthermore, colonic fermentation has been found to decrease post-prandial blood glucose response by as much as 15% amongst a healthy sample 21 . Increased colonic fermentation associated with consumption of FODMAP-dense foods indicates that FODMAPs have the potential to effect blood glucose response in a manner that is independent of glycemic index 12 . However, a possible prebiotic effect associated with FODMAP specific interventions has not been examined.
Increased colonic fermentation has also been linked to decreases in subjective appetite ratings. Supplementation of fructooligosaccharides has been found to promote expression of GLP-1, which leads to reduced ratings of subjective hunger in both clinical and free-living settings 22,23 . Furthermore, increased colonic fermentation has been associated with decreased energy intake 24 .
Research, to this point, has indicated that increases in colonic fermentation of carbohydrates will reduce post-prandial blood glucose response. However, these studies involved test meals or supplementation designed to deliver a targeted amount of oligosaccharides or soluble fibers. The ability of a FODMAP specific dietary intervention to elicit this effect has not been examined. Furthermore, a possible prebiotic effect associated with increased FODMAP intake, in a free-living setting, has not been examined. The present study examined how implementation of low-and high-FODMAP diets, in a free-living setting, would impact fasting and post-prandial glycemic response amongst healthy participants. Additionally, this study examined how changes in dietary FODMAPs would affect subjective hunger, satiety and prospective consumption, in both fasting and post-prandial states. Total FODMAP intake and fasting breath hydrogen were analyzed as a means to determine dietary compliance.

Materials and Methods:
Study Design: The study utilized a randomized, single-blinded, cross-over design that compared low and high-FODMAP diet-interventions in a free-living setting. Study design and laboratory procedures are outlined in figure 1. This study consisted of five separate data collection days. Initial data collection (i.e. prior to any dietary intervention or outcome testing) consisted of screening, completion of informed consent, instruction regarding study protocol, anthropometrics, and body composition assessment (via BODPOD). Baseline data collection took place on a Tuesday and required that the subject arrive at the laboratory following a 10-hour fasting period.
Fasting anthropometrics, blood glucose, breath hydrogen and subjective appetite ratings were obtained. A high FODMAP test meal (described below) was then administered and post-prandial blood glucose response and subjective appetite were measured at 30 and 60 min post consumption of the test meal. A 24-hour dietary recall was taken using the Nutritional Data System for Research (NDSR). At the conclusion of the 1-hour test period, individuals were randomly assigned to follow either diet 1 (low-FODMAP) or diet 2 (high-FODMAP) for a period of three days. Subjects reported to the laboratory for post-intervention testing on the following Friday. The post-intervention laboratory protocol was identical to that of baseline testing. After post-intervention testing, subjects undertook an 11-day washout period and the protocol, with the alternative diet, was repeated beginning on the following Tuesday.
Order randomization started with a coin flip for the first subject, and subjects altered order thereafter.

Subjects:
All subjects participating in this study were recruited as a convenience sample from the student population of the University of Rhode Island. Exclusionary criteria included gastrointestinal disorders such as: celiac disease, IBS, lactose or gluten intolerance, diverticular disease, colitis such as Cohn's disease or ulcerative colitis and stomach ulcers. Additional exclusion criteria included currently following a weight loss diet, food allergies, smoking, pregnancy or lactation, diabetes, adrenal disease, kidney or bladder problems, a thyroid disease or currently taking any appetite suppressant medication. Subjects were advised to keep physical activity constant throughout the course of the study. The study was approved by the Institutional Review Board of the University of Rhode Island and subjects provided written informed consent prior to participating.

Blood Glucose Analysis:
Blood glucose was analyzed at three separate time points during each laboratory session. Capillary blood from subjects fingers was sampled at 0 minutes

Assessment of Subjective Hunger, Satiety and Prospective Consumption:
Subjective appetite was assessed using 10-cm visual analog scales (VAS).
During laboratory sessions subjects completed VAS questionnaires regarding satiety, hunger, and prospective food consumption. VAS has been determined to be an effective method for assessing these outcomes 25 .

Assessment of FODMAP Intake:
FODMAP intake was assessed through 24-hour recalls utilizing NDSR.  27 . A healthy individual can consume between 25-50 grams of lactose before malabosrption can occur and was excluded from analysis 28,29 . Thus total FODMAP intake was considered fructose consumed in excess of glucose, and sugar alcohols.
Oligosaccharide intake is not assessed by NDSR databases and to our knowledge is not available in any nutrient database reflecting the United States food supply.  31 . Samples were collected in duplicate and the results were averaged.

Anthropometrics:
All anthropometric measurements were obtained at the beginning of each laboratory session, after the subject had undergone a 10-hour overnight fast and had voided bladder. Height, weight, and waist circumference were all measured and BMI was calculated. The protocol utilized for collection of anthropometric data has been described elsewhere 32 .

High-FODMAP Test Meal:
The test meal was designed to mimic free-living conditions.

Blood Glucose:
Two subjects were excluded from biochemical analysis due to abnormal blood glucose values. Figure 3 compares blood glucose response during the low-and high-FODMAP conditions. Following the high-FODMAP intervention, there was a non-

Summary:
This study is novel because it was the first study to examine a possible prebiotic effect associated with a free-living, high-FODMAP diet. The purpose of this particular study was to examine how changes in dietary FODMAP consumption, amongst a healthy sample, in a free-living setting, would affect blood glucose and subjective appetite ratings in both fasting and post-prandial conditions. The results of this study indicate that there was limited dietary compliance during the high-FODMAP intervention. This limited compliance resulted in non-significant differences regarding fasting breath hydrogen. There were no significant differences regarding blood glucose concentrations as a result of the dietary interventions. Partial eta squared was calculated and indicated that there was a small effect size between the two interventions (0.155). However, the negative correlation seen between breath hydrogen and blood glucose is suggestive of a prebiotic effect.
To examine a possible prebiotic effect associated with dietary FODMAPs individuals with the highest total FODMAP intake were analyzed. Significant decreases regarding blood glucose response were seen in the sample of compliant individuals. No changes significant regarding subjective appetite were seen in the sample compliant with the high-FODMAP diet.

Effect of FODMAP intake on blood glucose response:
Fermentation of dietary carbohydrates by colonic microbiota has been found to influence blood glucose response through increased production of short-chain fatty acids (SCFA). SCFAs bind to L-cells lining the intestinal epithelium resulting in increased production of a variety of gut-peptide hormones, most notably glucagonlike-peptide 1 38 . Previous studies have shown that supplementation of fructooligosaccharides can increase concentrations of GLP-1 in the proximal colon by 50% 39 . Binding of GLP-1 to beta cells increases expression of glucose transporters making the beta-cell more "sensitive" to the presence of extracellular glucose 40,41 . An increase in GLP-1 has been associated with increased activity of beta-cells in the presence of glucose 40,42 . Experimentally, increased endogenous production of GLP-1 has been inversely associated with blood glucose response amongst a healthy sample 20 .
The present study analyzed blood glucose across the sample of 16 participants; however, no significant differences between the two dietary interventions were seen.
This was likely due, in part, to poor dietary adherence under high FODMAP conditions. Amongst the sample of 16 subjects, the low FODMAP dietary intervention was successful in decreasing FODMAP intake from baseline assessment.
Under the high FODMAP intervention, FODMAP intake increased by 4.52 grams over the low FODMAP diet. While this was a statistically significant difference, this discrepancy regarding FODMAP intake between the two diets was likely not large enough produce a prebiotic effect.
Dietary compliance amongst the sample was also considered. Individuals with the highest FODMAP intake throughout the high-FODMAP intervention (n=8) were analyzed. Reanalysis of blood glucose for this sample showed a significantly reduced blood glucose response. Furthermore, this sample showed reduced post-prandial blood glucose response 30 minutes after administration of test meals. Previous studies show similar changes in post-prandial glycemic responses; however, these interventions were associated with considerably more FODMAP intake 21,22 .
Brighenti et al. 12 showed significantly reduced glycemic response amongst a sample consuming 5 grams of lactulose 12 . However it should be noted that these studies involved supplementation of FODMAPs. In order to achieve target FODMAP intake,

Determination of sample compliant with high-FODMAP diet:
Assessment of FODMAP intake for dietary compliance was performed in accordance to previous research 4, 33 . The NDSR database utilized for quantification of FODMAP intake did not assess dietary oligosaccharides consumed. Assessment of total FODMAP intake included fructose, lactose, and sugar alcohols. Fructose and lactose are considered to be conditional FODMAPs due to the variation regarding their absorption amongst healthy populations. Fructose, for example, is absorbed through two transport mechanisms: the GLUT 2 and GLUT 5 transporters. The GLUT 5 transporter is specific for fructose, has a high affinity for the monosaccharide, however, its ability to absorb glucose is limited 43 . The GLUT 2 transporter absorbs fructose in conjunction with glucose; therefore dietary fructose is only completely absorbed when equimolar concentrations of glucose are present 44,45 . Thus in order to be considered a dietary FODMAP, fructose intake had to exceed that of glucose 4,33 .
Lactose was not considered a FODMAP for the purposes of this study due to the selection criteria allowing only for individuals who had no diagnostic history of lactose intolerance and considered themselves able to consume lactose.

Breath Hydrogen:
In addition to SCFAs, hydrogen is another common byproduct of fermentation of malabsorbed carbohydrates. Hydrogen gas, produced in the colon, is absorbed through the walls of the intestine, entering the bloodstream, and is subsequently excreted as a component of exhaled breath 46 . Microbial fermentation of carbohydrates is considered the only means through which this "breath hydrogen" can be generated 29 . Breath hydrogen testing is a reliable method of measuring the extent to which carbohydrates are being malabsorbed and fermented 8,33,47 48 . Importantly, this study did find an inverse relationship between fasting breath hydrogen values and blood glucose response indicating a close relationship between increased colonic fermentation and glycemic response. Furthermore, these findings are consistent to that of previous research 20 .

Effect of intervention on subjective appetite:
Gut peptide hormones have also been shown to impact subjective appetite. For example GLP-1 has also been shown to bind to afferent nerve fibers of the central nervous system resulting in increased feelings of satiety 40,42 . Intravenous infusion of GLP-1 in a healthy human sample resulted in increased feelings of "fullness" and satiety that were not seen in the control group (p<0.03) 49 . Peptide PYY is another gut peptide hormone found to impact subjective appetite. Obese individuals are subject to reduced endogenous PYY concentrations, and increases in endogenous PYY has been found to attenuate appetite 50 . Cani et al. 22 showed increased colonic fermentation associated with FOS supplementation resulted in increased GLP-1 and PYY, and subsequent reductions in subjective hunger after a standardized test meal 22 . A recent study regarding supplementation of fructans, in a free-living sample, reduced subjective hunger ratings in a dose dependent manner (p<0.03) 23 . In the present study, there was no difference between the two dietary interventions regarding subjective appetite ratings perhaps due to insignificant dose.

Strengths and limitations:
The crossover design of this study was a major strength since each individual served as his or her own control and eliminated possible inequities in experimental and control groups that may exist in between group experimental designs. All outcomes in this study (blood glucose, cholesterol, and subjective appetite) were measured utilizing validated instrumentation.
Possible limitations of this study include the use of NDSR to analyze FODMAP intake. The inability of NDSR to quantify oligosaccharide consumption in our sample limited our ability to access dietary compliance. Future studies may consider alternative methods of assessing FODMAP intake 51 . Further limitations was our small sample of healthy participants, which limits the generalizability of our findings. Further work my consider utilizing subjects with, or at risk of type two diabetes.

Conclusion:
The dietary intervention, utilized throughout this study, was unsuccessful in increasing FODMAP intake sufficiently to produce a prebiotic effect in the sample of 16 healthy young adults. However, when examining data from individuals consuming greatest quantities of FODMAPs during the high FODMAP period of the intervention, a potential prebiotic effect was encountered as seen in reduced postprandial blood glucose response. This indicates a possible prebiotic potential of FODMAPs consumed in a diet of free-living individuals. However, longer-term studies, with larger compliant samples are needed to confirm these results.

A. REVIEW OF THE LITERATURE
Overview: Approximately 100 trillion bacterial cells colonize the Human colon. 57 . (apples and pears), as well as honey 63 . Fructans are also commonly seen in grains and pasta products most notably wheat pasta, gluten-free pasta, and quinoa pasta 1 . The ability of the small intestine to absorb dietary FOS is severely limited 64 .
Experimental evidence indicates that up to 89% of ingested fructans reach the colon intact 64 . Thus the energy production associated with fructans is estimated to be 9.

Malabsorption of FODMAPs:
Malabsorption of macronutrients is regarded as the inability of the digestive system to enzymatically hydrolyze and absorb nutrients in the small intestine. On one hand, carbohydrates associated with α-glycosidic linkages are readily absorbed by the human body 70,71 . Salivary and luminal hydrolase enzymes readily cleave these bonds allowing for absorption of these carbohydrates through transport proteins located in the first mucosal membrane of the small intestine 70 . These hydrolase enzymes are unable, however, to hydrolyze the β-glycosidic linkages that are associated with FODMAPs, thus inhibiting digestion and absorption of these carbohydrates 70 .
The lumen of the small intestine presents a substantial barrier regarding the rapid diffusion of large water-soluble molecules into enterocytes 72  Furthermore, increases in FODMAPs found in ileostomy effluent were associated with a 22 % (95% CI, 5%-39%) increase in water content, indicating that malabsorbed carbohydrates are osmotically active 5,50 . This analysis confirms the ability of a high FODMAP diet to exert an osmotic effect on the small intestine and establishes a mechanism through which these carbohydrates can affect gastrointestinal tolerance.

Influence of FODMAPs on Orocecal Transit:
The ability of dietary FODMAPs to exert an increased osmotic effect can have important implications regarding orocecal transit. The ability of FODMAPs to draw water into the lumen of the intestine results in increased peristaltic activity increasing the rate at which food travels through the gastrointestinal tract 73 . Ingestion of 25g of fructose and 5 g of sorbitol was found to have a significantly higher rate of intestinal transit when compared to control (P= 0.0033) 73 . Furthermore, the length of carbohydrate polymers was found to significantly effect orocecal-transit. Short chain carbohydrates (degree of polymerization >10), that are malabsorbed, have been found to have reduced orocecal transit times when compared to longer chained carbohydrates (degree of polymerization <10) 74 . This shows that FODMAP consumption can alter the function of the small intestine and deliver ingested nutrients to the colon at a higher rate than longer chained, less osmiotically active carbohydrates.

Rapid Fermentation of Malabsorbed FODMAPs:
Malabsorbed carbohydrates are subsequently deposited in the proximal colon where they serve as substrate for microbial metabolism. Colonic microbiota utilize fermentation reactions to metabolize FODMAPs, which is characterized by increases in hydrogen gas formed in the colon. In conditions associated with low dietary FODMAP consumption, production on hydrogen gas by colonic micro flora is low.
However, increases in dietary FODMAPs will result in measurable increases in hydrogen production 75,76 . Hydrogen gas that is formed through this process is absorbed through the wall of the colon where it enters the blood stream. Once it enters the blood stream, the hydrogen gas is subsequently excreted through the lungs 75,76 .
Thus any changes in breath hydrogen concentrations can be attributed to colonic fermentation 75,76 .
Due to their short-chain length and ability to exert an osmotic effect, dietary

Role of FODMAPs in IBS:
The poor digestion, osmotic effect and high rate of bacterial metabolism that

Potential prebiotic effect of FODMAPs:
There is a multitude of different species and strains of bacteria that colonize the human colon. Some species of microbiota such as bifidobacteria and lactobacilli are beneficial to the overall health of the host organism yet others (eg. Clostridium) can negatively affect the overall health 80  The ability of FOS to exert a prebiotic effect has been studied rather extensively. FOS supplemented in bacterial culture of 21 different strains of bifidobacterium was associated with significant bacterial growth. Almost all of the strains of bifidobacterium were able to grow on a medium containing FOS, while only 8 strains were able to show significant growth on mediums containing the longer chain inulin 82 . Fructooligosaccharides have also proven to be an effective prebiotic in human models as well. Fecal bacterial concentrations, in humans, were found to have a dose-dependent relationship regarding oligofructose supplementation (p<. 009) 83 .
This indicates that supplementation of FOS can be advantageous regarding cultivation of colonic bacterium that are regarded as beneficial for human health.
While not studied as extensively as FOS, GOS supplementation, in healthy human populations, has also been associated with a significant probiotic effect.
Supplementation of 10 grams of GOS throughout a 21 day intervention period showed a significant increase in breath hydrogen accompanied by increases in bifidobacterium 84 . More recently, supplementation of 10grams of GOS for a 10 day period was found to have significant increase concentrations of colonic microbiota (P<0.0001) 84 . These results were not seen with longer chain starches and fibers indicating that the low degree of polymerization associated with GOS can more effective prebiotics than longer chain fibers 83 .
Due to poor gastrointestinal tolerance, polyols research regarding a possible prebiotic effect is lacking. Polyols, such as sorbitol, are associated with similar chemical properties as other FODMAPs such as GOS or FOS, thus a possible prebiotic effect associated with colonic fermentation of polyols cannot be ruled out. In vitro analysis has shown that supplementation of sorbitol to bacterial media results in increased bacterial proliferation, most notably increases in lactobacilli, which are largely considered to a beneficial species of colonic microbiota 85 . Furthermore, sorbitol supplementation in rats has been associated with a prebiotic effect as well.
Supplementation of sorbitol over a 16-day period in rats resulted in significant increases in lactobacillus 86 . While a direct link between polyol consumption and a prebiotic effect in humans has yet to be established experimentally, thus this area of study warrants further research.

Prebiotics and Short-Chain Fatty Acids and Gut peptide hormone production:
Prebiotics are considered to be beneficial to the overall health of the host organism through the ability of these carbohydrates to cultivate the growth of favorable species of colonic microbiota. As previously stated, high levels of bifodobacterium and lactobacilli are associated with health. One of the main hypothesis through which these species of colonic microbiota are able to elicit this beneficial response is through production of short chain fatty acids (SCFA) 87,88 .
Short-chain fatty acids are a byproduct of anaerobic metabolism of dietary carbohydrates. Experimental evidence indicates that SCFA production through bacterial fermentation is linked increased serum concentrations of gut peptides such as glucagon-like peptide 1 and peptide YY 89 . Increases in these peptides have been associated with enhanced insulin sensitivity, increased feelings of satiety, and reduced energy intake 43 .
Glucagon-like peptide 1 (GLP-1) is known to influence a wide variety of metabolic processes throughout the body 44 . GLP-1 is released by L-cells located in the intestine in response to food intake 44 . GLP-1 has shown the ability to confer insulin sensitivity through binding to pancreatic beta cells 43 . Binding of GLP-1 to beta cells increases expression of glucose transporters making the beta-cell more "sensitive" to the presence of extracellular glucose 43,44 . An increase in GLP-1 has been associated increased activity of beta-cell in the presence of glucose 43,45 .
Furthermore, GLP-1 has also been shown to bind to afferent nerve fibers of the central nervous system resulting in increased feelings of satiety 43,45 . GLP-1 is an important modulator of metabolic homeostasis and has potential to promote decreased energy intake and weight loss 43,45 .

Glycemic Index, Colonic Fermentation and Blood Glucose:
Glycemic index (GI) is defined as "blood glucose-raising potential of a standard quantity of carbohydrate, compared with a glucose control" 93,94  One of the most important health benefits associated with consumption of LGI foods is the ability of these foods to promote "a second meal effect." A concept, originally described by Jenkins et al. 95 and Wolever et al 13 states that the glycemic index of an initial meal is a determinant of the postprandial physiological response at subsequent meals [13][14][15]95 . During a one day test period, healthy volunteers, who were administered a LGI "breakfast" were found to have diminished insulin and post prandial blood glucose responses after administration of a HGI lunch 4 hours later 15 .
A follow-up study found a similar relationship between glycemic index and postprandial blood glucose response at subsequent meals 14 . While these studies were not able to establish a relationship between colonic fermentation and improved glucose tolerance, LGI foods containing large amounts of poorly absorbed carbohydrates have the potential to improve glucose tolerance, a concept supported by subsequent research 14,15 .  39 . Subjective ratings of satiety were found to be positively correlated with breath hydrogen (r=-0.27; p<0.01) 21 . These studies establish a strong connection between colonic fermentation, thus increased microbial metabolism of poorly digested LGI foods, and improved postprandial responses in a healthy adult population.
More recently, a study was conducted examining the possible second meal effect associated with consuming a meal of brown beans. Brown beans are not only considered to be a low GI food, they are also associated with over 8 grams of soluble fiber, 6 resistant starch, and 3 grams of raffinose (a common galactooligosaccharide) per portion of brown beans 20 . Supplementation of these brown beans, amongst a healthy population, proved to be readily metabolized by colonic microbiota. Breath hydrogen, utilized as an indicator of colonic metabolism, increased 141% compared to the control (p<0.01) 20 . These increases in colonic fermentation were also associated with subsequent increases in the short-chain fatty acids proprionate (16% p<0.05) and isobutyrate (18%; p<0.01). In addition, satiety mediating hormones were increased as Lactuose is a synthetically derived disaccharide that is malabosrbed and subsequently fermented by colonic microbiota 12 . Results showed that the LGI and HGI-Lactulose interventions resulted in similar postprandial blood glucose responses when compared to the HGI meal. Improved glucose tolerance was noted in both interventions and significant increases in breath hydrogen were seen (p<0.001 for both LGI and HGI-Laculose). This analysis shows that colonic fermentation of carbohydrates, independent of glycemic index, can improve subsequent glycemic response post meal 12 .

Colonic Fermentation and Subjective Hunger and Energy Intake:
The ability of dietary FODMAPs to reduce postprandial insulin and glucose responses is important regarding their ability to induce prolonged periods of satiety 96 .
Meals that are high in glycemic index are associated with a sharp increase in blood glucose immediately following meal consumption followed by a drastic decrease in blood glucose 2 hours following the meal 96 . This sharp decline in blood glucose levels has been linked to feelings of hunger and has been seen to immediately precede meal initiation. A meal associated with a low glycemic index does not induce the drastic increases in blood glucose that are seen with HGI meals providing prolonged feelings of satiety. HGI meals were found to increase subjective appetite scores by as much as 44% amongst a population of obese females 96

Metabolic Functions of AMPK:
AMPK is a serine/ threonine kinase and is an important regulatory protein that acts to modulate cellular energy homeostasis 99,100 . Increases in AMPK activity is associated with low cellular energy concentrations (high AMP/ATP ratio). Thus AMPK acts to increase cellular glucose concentrations through activation of GLUT 4 transporters (increasing cellular glucose uptake) and has also been linked to upregulation of cellular glycolysis through activation of 6-phosphofructo-2 kinase and fructose-2,6-bisphosphatase 99,100 . AMPK helps to maintain cellular homeostasis, in times of energy depletion, through up-regulation of catabolic (energy producing) processes.
AMPK also inhibits anabolic processes (energy consuming pathways) from occurring, most notably hepatic cholesterol synthesis. Research indicates that AMPK has the ability to inhibit cholesterol synthesis in a similar fashion to statins 101 .
Cholesterol is synthesized from acetyl-CoA subunits through a complex metabolic pathway in which HMG-CoA reductase catalyzes the rate-limiting reaction 102,103 .
HMG-CoA reductase is subject to enzymatic inhibition through phosphorylation of the Serine-872 residue 103 . This reaction, catalyzed by AMP-activates protein kinase (AMPK) inhibits the binding of the NADH cofactor to the protein, rendering the protein non-functional 103 .

Short Chain Fatty acids and AMPK activity:
Increases in serum concentrations of SCFA have been associated with increases in the activity of hepatic AMPK 102 . While the mechanism through which SCFAs promote the activity of AMPK has yet to be elucidated, it has been hypothesized that SCFAs increase the AMP/ATP ratio resulting in the subsequent activation of AMPK 102 . Research by Kawaguchi et al. 104 and Sakabibera et al. 105 suggest that supplementation of acetate in mice resulted in increased activity of hepatic AMPK. These studies indicated that SCFAs have the ability to directly increase catalytic activity of AMPK 104,105 .
Fushimi et al 106 showed that supplementation of SCFA can be directly related to decreased rate of cholesterol synthesis. This study examined the effect of supplementation of acetate in a sample of rats. The analysis showed that supplementation of acetate was associated with decreased hepatic concentrations of malonyl-COA indicating inhibition of HMG-COA reductase 106 . This analysis indicates that increases in serum concentrations of SFCA can prove advantageous regarding reduction in cholesterol synthesis.

Short Chain Fatty Acids and Bile synthesis:
An alternate mechanism through which SCFAs can impact serum cholesterol levels is through increased hepatic bile synthesis. Bile acids, synthesized in the liver, are excreted after a meal and function to facilitate the digestion and absorption of lipids 44,107 . While bile is composed of a variety of organic compounds, bile acids and free cholesterol are considered to be a main component 44

Colonic fermentation, SCFAs, and cholesterol synthesis in Humans:
In the context of a normal human diet, increases in SCFA are achieved through

Overview:
The methodology for this particular study was developed in the Energy Balance Laboratory in the department of Nutrition and Food Sciences, at the University of Rhode Island. Data collection for this study took place during the spring/summer of 2013. This study utilized a randomized, single-blind, crossover design to measure pre-and post-prandial physiological responses of healthy individuals exposed to both high and low FODMAP diets. After baseline data was collected, participants were carefully instructed on the incorporation of high or low diet for a period of three days.

Post-Intervention Assessment:
Post-Intervention testing consisted of visits three and five and took place after the subject had been following the dietary intervention for a period of three days.
Since baseline assessment took place on a Monday, post-intervention testing took place on Friday. Laboratory protocols, for post-intervention assessment, was identical to that of baseline assessment. Subjects received a total of eighty dollars for their participation in the study. Twenty dollars was paid after session three and sixty dollars was paid after the fifth and final visit.

High-FODMAP Test Meal:
The test meal was designed to mimic real world conditions. The 25g of FODMAPS associated with the test meal has been found to induce increases in breath hydrogen concentration indicating carbohydrate malabsorption 9

Educational Material:
To assist with dietary adherence, each participant received educational material regarding the assigned diet. The educational material came in the form of a booklet that assisted the participant in choosing foods included in their particular diet conditions. The educational material contained "foods to eat" and "foods to avoid" for each food group (see sample in Appendix G). The educational material utilized during this study was created specifically for the purposes of this study utilizing previous research 2, [33][34][35] . Educational materials regarding low FODMAP conditions will emphasize the reduction in consumption of fructose, lactose, fructans, and polyols, as these are common in the western diet 2  will then be calculated utilizing these two measurements. Waist circumference will be measured using a standard tape measure with tensometer. Measurement will be obtained by placing the tape measure around the participant's waist at the level of the umbilicus. If there is greater than 0.5 cm difference, then the measurement will be repeated until two measurements are within this range.
Protocol utilized for collection of anthropometric data has been described elsewhere 33 .

Sample Size:
Sample size calculations were performed based on differences in blood glucose response seen in a previous study by Nilsson et al 39 . This study found that peak blood glucose concentrations decreased 1mmol/L (18 mg/dl) from control to experimental conditions. Calculations show an effect size of 3.33. All calculations were based on an alpha of 0.05 and power of 0.80, and were completed using G* Power (version 3.1.7). According to these calculations, a 8-person sample size would be adequate to achieve sufficient power. Therefore, the proposed sample size of 16 will provide appropriate statistical power for measuring changes in blood glucose response in healthy individuals.

Visit 4 Scheduling
___ Inform subject that they will now have an eleven-day washout period where they should not follow any specific diet (eat what they usually eat) ___ Inform subjects that they must refrain from eating or drinking anything except water before reporting to the lab for their next visit Visit 4 must be scheduled eleven days from now on Tuesday and after a 10 hour fast. That being said, the date of the participant's next appointment will be:

CONSENT FORM FOR RESEARCH
You have been invited to take part in a research project described below. The researcher will explain the project to you in detail. You should feel free to ask questions. If you have more questions later, Kathleen Melanson, the person mainly responsible for this study, will discuss them with you (Energy Balance Lab, 310 Ranger Hall, 401-874-2067). You must be at least 18 years old to be in this research project. You will also need to be available for two Tuesday and two Friday mornings for testing.

Description of the project:
You have been asked to participate in a research study designed to test the acceptability of and physiological responses to the manipulation of dietary carbohydrate sources.
What will be done: Following an initial assessment visit, all study volunteers will follow two specific diets for three days each, with 11 days of your normal eating inbetween: • You would follow the first diet for three days between the second lab and third lab visits. • During the 11-day period between the third and fourth lab visits, you would not be asked to follow any specific diet; you would just return to your normal diet. • You would follow the second diet for three days between the fourth and end the fifth lab visits, which marks the end of the study.
Total time in the lab will amount to about 8.5 hours: 30 minutes for first visit, and about two hours each for the other four visits. You will note below that the two-hour visits include a one-hour wait between consumption of a test breakfast and the second round of measurements.
While some of this wait period will be used to perform a 24-hour dietary recall (visits 2-5) and give instructions for your assigned diet (visits 2 and 4), these activities are not likely to consume the whole 1 hour waiting time. For that reason, it is recommended that you bring homework or some form of activity to occupy yourself between measurements.
Your total compensation for completing the study will be $80. You will receive $20 following the third lab visit, after completion of the first test diet. You will receive the remaining $60 following the fifth and final lab visit, after completing the second test diet and concluding your participation in the study.
• First Visit (~30min) o We will ensure your eligibility for this study by verifying your age and the absence of exclusionary criteria. o We will explain to you what this study entails and what will be expected of you as a participant in this study. o After you have had a chance to ask any questions, you will be given a consent form to fill out. o Upon completing the consent form and agreeing to partake in the study, you will have your body composition measured using the BodPod. You will be asked to sit inside the BodPod machine and comfortably rest for 2-5 minutes while your body fat percentage is estimated. This machine is a large eggshaped capsule, with a hatch that has an internal 'panic button' in case you want to open the hatch while you are in there. For this test, we will ask you to bring a bathing suit to wear while you are in the BodPod. This simply minimizes the chances that bulky clothing will affect the reading. We will provide you with a swimming cap to place over your hair; this also minimizes the chances that air caught around your scalp would affect the reading. Your body fat is estimated in this fashion by calculating the amount of air you displace inside the known area of the BodPod. This test should take less than 20 minutes.
• Second Visit (~3hrs) o You will report to the lab following an overnight (10-hour) fast.
o Your height, weight, and waist circumference measures will be taken. o You will be asked a short series of questions regarding your appetite. o You will have a small amount of blood taken by fingerstick to measure your blood glucose concentration. o You will have your breath hydrogen measured by exhaling into a small bag. o You will eat a test breakfast. o 30 minutes following the breakfast, your blood glucose will be measured again and you will again be asked to answer questions regarding your appetite. o two hours following the breakfast, your blood glucose and breath hydrogen measurements will be taken again, and you will again be asked to answer questions regarding your appetite. o During the two hours between finishing the breakfast and the second round of tests, you will be asked to recall what you have had to eat and drink in the past 24 hours. o Following the 24-hour recall, you will be given instructions pertaining to the diet to which you have been assigned for the first leg of the experiment. You will also receive reference materials you can take with you to help you adhere to the assigned diet.
You will be asked follow the assigned diet to the best of your ability for three days, beginning after you leave the lab up until 10 hours before the third lab visit. On each of these days you will complete short questionnaires about your appetite, how you feel, and your opinion of the diet. For the 10 hours before the third visit, you will be asked to again refrain from eating or drinking anything except water before reporting to the lab for the third visit.
• Third Visit (~2hrs) o You will report to the lab following an overnight (10-hour) fast. o Your height, weight, and waist circumference measures will be taken. o You will be asked a short series of questions regarding your appetite. o You will have a small amount of blood taken by fingerstick to measure your blood glucose concentration.
o You will have your breath hydrogen measured by exhaling into a small bag. o You will eat a test breakfast. o 30 minutes following the breakfast, your blood glucose will be measured again and you will again be asked to answer questions regarding your appetite. o Two hours following the breakfast, your blood glucose and breath hydrogen measurements will be taken again, and you will again be asked to answer questions regarding your appetite. o During the two hours between finishing the breakfast and the second round of tests, you will be asked to recall what you have had to eat and drink in the past 24 hours.
Following the third visit, you will receive $20 in compensation for completing the first test diet. You may retain this payment even if you choose to withdraw before completion of the study. There will be an 11day period during which you will not be asked to follow any specific diet.
• Fourth Visit (~2hrs) will include the same procedures as the Second Visit.
You will be asked follow the next assigned diet to the best of your ability for three days, beginning after you leave the lab up until 10 hours before the third lab visit. On each of these days you will complete short questionnaires about your appetite, how you feel, and your opinion of the diet. For the 10 hours before the third visit, you will be asked to again refrain from eating or drinking anything except water before reporting to the lab for the fifth visit.
• Fifth Visit (~2hrs) will include the same procedures as the Third Visit.
Following the fifth visit, you will have completed your participation in this study. You will receive an additional $60 at the end of the fifth visit, for a total of $80 in compensation for completion of the study.

Risks or discomfort:
There are no known risks for the following procedures: questionnaires, consumption the manipulated carbohydrate diets, measures of height, weight, waist circumference, body composition, breath hydrogen, food intake and appetite questionnaires.
The finger prick can result in some slight, short term pain and discomfort. Even though trained, experienced personnel will perform the blood draw using sterile technique, it is possible that minor bruising and infection may occur.
If during the course of the analysis, any incidental findings emerge that indicate a health risk to you (such as high fasting glucose levels), you will be informed and will be advised to consult with your personal physician.

Benefits of this study:
This study will help to determine the effects modified carbohydrate diets on several physiological factors relevant to clinical and therapeutic applications. The direct benefits to you include learning about your body composition, blood glucose, and diet. Upon completing the first test diet you will receive $20.00 in compensation. Upon completing the second test diet you will receive $60.00 in compensation, for a total of $80.00 for completion of the study.

Confidentiality:
Your participation in this study is confidential. All of your information will be coded by an identification number after all of your data have been collected. None of the results of this study will identify you by name. The document linking your name and identification number and all data collected will be stored on password-protected computers and in locked file cabinets within the locked lab of the investigator. Access will be limited to study investigators. Federal regulations require that data and signed consent documents be kept for three years following completion of the study. The researchers and the University of Rhode Island will protect your privacy, unless they are required by law to report information to city, state or federal authorities, or to give information to a court of law.
Otherwise, none of the information will identify you by name.
In case there is any injury to the subject: Decision to quit at any time: The decision to take part in this study is up to you. You do not have to participate. If you decide to take part in the study, you may quit at any time. Whatever you decide will in no way penalize you. If you wish to quit, you simply inform Dr. Kathleen Melanson at (401) 874-4477 of your decision.