Mechanism of Microbiome-induced Insulin Resistance in Humans (Aim 1)
Phase: PHASE2
Conditions: Insulin Sensitivity
Sponsor: The University of Texas Health Science Center at San Antonio

COVER PAGE FOR PROTOCOL AND STATISTICAL ANALYSIS PLAN 
Official Study Title: Mechanism of microbiome‐induced insulin 
resistance in humans (Aim 1) 
NCT number: NCT02124759 
IRB Approval Date: 08/16/2013 
Unique Protocol ID: HSC20130459H 

 
1. RESEARCH PLAN 
A) SPECIFIC AIMS 
Insulin resistance in peripheral tissues (i.e. skeletal muscle) is one of the earliest and most significant 
abnormalities in the pathogenesis of type 2 diabetes (T2DM).  However, the molecular basis for the insulin 
resistance of T2DM is not fully understood.  The human gut hosts an enormous number and variety of 
microorganisms, including at least 1014 bacteria.  An increasing number of animal studies suggest that this 
microbial “organ”, also known as the microbiome, is a causative factor in the insulin resistance of obesity and 
T2DM, and may represent an entirely new therapeutic target against these conditions.  These studies suggest 
that high fat ingestion, obesity, and T2DM, favor a microbiome profile which enhances production and 
gastrointestinal permeability of lipopolysaccharide (i.e. metabolic endotoxemia).  Lipopolysaccharide (LPS or 
endotoxin) is a component of the outer membrane of gram negative bacteria cell walls which induces an 
inflammatory response by activating the cell surface receptor toll-like receptor-4 (TLR4).  Despite accumulating 
evidence from animal studies suggesting that intestinal microbiota and metabolic endotoxemia could play a key 
role in the pathogenesis of insulin resistance, obesity, and T2DM, the relevance of the intestinal microbiome 
and metabolic endotoxemia on human metabolic disease remains unknown.  Thus, the goals of this study are to 
(i) determine the role that intestinal microbiota and metabolic endotoxemia play in the pathogenesis of insulin 
resistance and T2DM in humans; and (ii) examine the cellular mechanisms by which metabolic endotoxemia 
causes insulin resistance in human muscle.  Specifically, we propose the following Specific Aims:    
Aim 1) To determine the effect of high fat consumption on the intestinal microbiome, metabolic 
endotoxemia, and insulin action, in lean normal glucose tolerant subjects.  We will test the hypothesis that a 
high fat diet given to lean, normal glucose tolerant subjects will modify gut microbiome composition and 
enhance intestinal permeability, which will increase plasma LPS concentration, induce an inflammatory 
response in peripheral tissues (skeletal muscle), and impair insulin signaling and sensitivity.  Also we will test 
the hypothesis that the inflammatory response and insulin resistance caused by high fat ingestion can be 
ameliorated by administering (i) a synbiotic (bifidobacterium and oligofructose) which protects the intestinal 
epithelial barrier and decreases intestinal translocation of LPS; and (ii) sevelamer, an agent which sequesters 
LPS in the gastrointestinal tract limiting its translocation into the circulation. 
Aim 2) To determine whether microbiome modulation and an experimental reduction in plasma LPS 
concentration improve inflammation and insulin action in insulin resistant (obese and T2DM) subjects.  
In this Aim we will test the hypothesis that lowering LPS concentration in the circulation will improve systemic 
(muscle) inflammation and glucose metabolism in insulin resistant (obese and T2DM) subjects by protecting the 
intestinal barrier with a synbiotic (bifidobacterium and oligofructose) or by sequestering LPS in the 
gastrointestinal lumen with sevelamer.     
Aim 3) To determine whether the LPS present in the sera from lean high fat fed, obese, and T2DM 
subjects mediates the insulin resistance, and whether this effect is mediated via TLR4.  In this Aim we will 
test the hypotheses that sera from lean high fat fed, obese, and T2DM subjects (from Aims 1 and 2) will induce 
an inflammatory response (activation of inflammatory signaling cascades) and impair insulin action in cultured 
primary myotubes obtained from lean individuals, and that the sera from these groups (lean on high fat diet, 
obese, and T2DM), when treated with LPS-reducing agents (synbiotic and sevelamer), will blunt the 
inflammatory response and insulin resistance. We will also specifically neutralize LPS in the sera from these 
subjects (lean on high fat diet, obese, and T2DM) using polymixin B, and will block TLR4 signaling in the 
myotubes with short hairpin (sh)RNA and a pharmacologic inhibitor (TAK242), to test the hypothesis that the 
inflammatory state and insulin resistance caused by the sera occurs via an LPS-TLR4-mediated mechanism. 
B. BACKGROUND AND SIGNIFICANCE 
The intestinal microbiota and metabolism.  A connection between the intestinal microbiota, energy 
homeostasis, and metabolism, and its role in the pathogenesis of obesity-related metabolic disorders, are 
increasingly being recognized. The human gut hosts an enormous number and variety of microorganisms (1).  
The genome size of this microbial “organ”, also known as the microbiome, exceeds the size of the human 
(nuclear) genome by two orders of magnitude, providing additional features and contributing to human 
1

 
physiological and metabolic diversity (2, 3).  In particular, the intestinal microbiota has been considered to be a 
possible causative factor in metabolic disorders such as insulin resistance and dyslipidemia, as well as a 
therapeutic target against these conditions (3).  Bacteria with beneficial effects such as Bifidobacteria and 
Bacteroidetes are decreased in obese and type 2 diabetic subjects (4, 5).  In addition, germ-free mice 
transplanted with intestinal microbiota derived from human fecal material have increased body fat relative to 
mice that do not have the humanized microbiome (6). Moreover, transplantation of microbiota from humans fed 
a high fat diet (as opposed to low fat diet) into mice leads to obesity (6).  These results suggest that intestinal 
microbiome composition has a significant effect on whole body energy homeostasis and fuel metabolism.   
Mechanism of glucose metabolism regulation by the microbiome.  The mechanism by which gut microbiota 
affect glucose metabolism and insulin action is not clear.  One hypothesis suggests that microbiome profiles 
enriched in certain bacterial groups/genus lead to enhanced production and gastrointestinal permeability of 
lipopolysaccharide (i.e. metabolic endotoxemia).  Lipopolysaccharide (LPS or endotoxin) is a component of the 
outer membrane of gram negative bacteria cell walls, which induces an inflammatory response by activating 
toll-like receptor (TLR)4.  Other potential mechanisms by which intestinal microflora can affect glucose 
metabolism in the host is through the production of substances which alter intestinal wall integrity (7, 8), energy 
extraction from food (9), energy expenditure, and appetite (10-13).  Activation of TLR5 by bacterial flagellin is 
another mechanism by which intestinal microbioma can affect glucose metabolism (14).  Bacterial 
peptidoglycans, which bind to and activate TLR2, have also been found to impair insulin action in muscle cells 
(15).  In addition, the gut microbiota affects the host’s capacity to extract calories from otherwise indigestible 
polysaccharides (16-18). 
TLR4 and downstream signaling. TLRs are a family of pattern recognition receptors that generate immune 
responses to pathogens by activating a cascade of pro-inflammatory events (6). TLR4, one of the best 
characterized TLRs, is highly expressed on the surface of immune cells, such as monocytes and macrophages, 
and insulin target cells, such as myocytes, adipocytes and hepatocytes. LPS is a potent agonist of TLR4 (7).  A 
soluble protein called LPS binding protein (LBP) binds LPS and regulates LPS-dependent responses.  Saturated 
free fatty acids (FFA) are also agonists of TLR4 (9, 10). TLR4 ligand binding leads to the activation of several 
pro-inflammatory kinases including the mitogen-activated protein kinases (MAPK); c-Jun N-terminal kinase 
(JNK), p38 and extracellular-signal related kinase (ERK), and the IκB kinase (IKK) complex, which results in 
the activation of transcription factors such as nuclear factor-κB (NFκB), c-jun, and AP-1 (12) and increased 
gene transcription of proteins such as tumor necrosis factor-α (TNFα), inducible nitric oxide synthase (iNOS), 
monocyte chemotactic protein (MCP)-1 and interleukin (IL)-6 (16, 17). Genetic or chemical inhibition of either 
JNK or IKKβ/NFκB can improve insulin sensitivity (18-20). Moreover, disrupted expression of TLR4 protects 
mice from developing inflammation and insulin resistance in response to chronic changes in dietary intake of fat 
(21, 22). These findings provide compelling evidence that insulin action is negatively regulated by TLR4. 
Metabolic endotoxemia and insulin resistance.  As mentioned above, accumulating evidence suggests that 
circulating intestinal-generated LPS (i.e. metabolic endotoxemia) could play an important role in the 
pathogenesis of insulin resistance. This emerging hypothesis is of particular interest to our laboratory since we 
recently observed that obese and T2DM subjects have elevated LPS concentration in plasma (Fig. 2), in 
association with decreased insulin sensitivity (M) (r=-0.46, P<0.005).  In support of our findings, ingestion of a 
high fat meal significantly increases circulating LPS level (19, 20). Unlike other situations of endotoxemia, such 
as sepsis where plasma LPS level may increase ~100-fold (21), in situations of insulin resistance LPS levels are 
only modestly increased by 2-3 fold, when compared to control subjects (22). Cani et al reported that, in mice, a 
high fat diet leads to increased plasma LPS concentration in association with alterations in the intestinal 
microbiota composition (22, 23).  Modulation of the intestinal microbiota composition with prebiotics reduced 
plasma LPS concentration and improved glucose intolerance in obese ob/ob and high fat fed mice (7, 23-26). 
The mechanism by which high fat diets increase plasma LPS concentration is not clear, but postulated 
mechanisms involve changes in the composition of the microbiome (7, 23-26), lipid- and or micriobial-induced 
damage of the intestinal epithelial barrier (i.e. increased permeability) (24), and post-prandial chylomicron 
formation which promote transport of LPS (27) (Fig. 1).   
2

 
In addition to the mouse 
studies described above, the 
role of LPS in triggering 
systemic inflammation also 
has been evaluated in 
healthy human subjects.  
Mehta et al. found that a 
low dose intravenous bolus 
of LPS increased TNFα and IL-6 concentrations and decreased insulin sensitivity (28).  Ghanim et al reported 
that a high calorie diet (high in fat and carbohydrates) caused a significant elevation of LPS in plasma, 
accompanied by increased mononuclear cell expression of TLR4 and NFκB (29).  The increases in LPS, TLR4 
and NFκB were absent after an American Heart Association-type meal, low in fat (29).  The same group 
performed a study to dissect the effect of high calorie diet (carbohydrate vs. fat), and found that a diet rich in 
fat, but not in carbohydrates, increased plasma LPS concentration and activated NFκB in circulating 
mononuclear cells (30).  In addition, a dietary survey conducted in 1015 subjects demonstrated that a high 
intake of fat, but not carbohydrates or protein, was directly associated with plasma LPS concentration (31). 
Collectively, the results described above suggest that caloric intake and nutrient composition (high fat) affect 
LPS concentrations, and that metabolic endotoxemia may play a key role in the pathogenesis of insulin 
resistance and T2DM.  However, the relevance of the intestinal microbiome and metabolic endotoxemia on 
human metabolic disease remains unclear.   Thus, the goals of this study are to (i) determine the role that 
intestinal microbiota and metabolic endotoxemia play in the pathogenesis of insulin resistance and T2DM in 
humans; and (ii) examine the molecular basis by which metabolic endotoxemia causes insulin resistance.  The 
results from this project could help to develop new interventions against insulin resistance and T2DM. 
C) PRELIMINARY RESULTS 
Plasma LPS concentration is increased in obese and T2DM individuals. We measured plasma LPS in 11 
lean (BMI=24.6 kg/m2, age=41 y, fasting glucose=93 mg/dl, M value=11.3 mg/kg.min), 9 obese, nondiabetic 
(BMI=31.5, age=47, glucose=92, M=8.1), and 10 obese, T2DM (BMI=34.2, age=48, glucose=145, M=4.2) 
subjects. Insulin sensitivity (M) was measured with a hyperinsulinemic (80 mU/m2.min) clamp.  As shown in 
Fig. 2, the plasma LPS concentration was significantly elevated in the obese 
and T2DM subjects.  It is important to note that a normal value for LPS 
concentration in human plasma has not been established and there is enormous 
variability in the concentration reported in normal subjects, from the 0.01 (32, 
33) to the 10 EU/ml (34) range, depending on the method employed, reagent 
manufacturer, and population studied.     
LPS causes insulin resistance in muscle in vitro.  We pre-treated cultured L6 
(rat) myotubes with 100 ng/ml LPS for 4 and 24 h, followed by stimulation 
with 100 nM insulin for 20 min.  LPS significantly reduced insulin-stimulated 
glucose (2DG) transport (Fig. 3).  
Pharmacologic inhibition of TLR4 protects against LPS-induced 
inflammation in vitro.  We examined the effect of TAK242 (Takeda 
Pharmaceuticals), an inhibitor of TLR4, on LPS-induced inflammation. L6 
myotubes were pre-incubated with/without 1µM TAK242 for 1 h, prior to 
stimulation with 100 ng/ml LPS for 1h. TAK242 completely blocked LPS-
induced JNK (Fig 4a) and p38 phosphorylation (Fig 4b).   
 
 
 
LPS causes insulin resistance in human muscle cells.  The data shown 
above clearly indicate that LPS impairs insulin action in rodent muscle cells.  Also we have tested the effect of 
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LPS in a primary human muscle cell culture system.  Myotubes derived from lean, nondiabetic subjects were 
treated with 10 and 100 ng/ml LPS for 1, 6, 12 and 24 h.  As shown in Fig. 5, LPS caused a robust increase in 
inflammatory gene expression in a dose- and time-dependent manner. To determine the metabolic consequence 
of the inflammatory response caused by LPS, human myotubes were treated with 100 ng/ml LPS for 1, 6, 12 
and 24 h, followed by insulin stimulation (100 nM for 20 min).  LPS reduced Akt (Fig. 6a), GSK3 (6b) and 
AS160 (6c) phosphorylation.  Furthermore, pre-treatment with 100 ng/ml LPS for 12 h impaired insulin-
stimulated glucose transport (Fig. 7).   
 
 
 
  
 
 
We have conducted experiments in muscle cells using lower (more clinically-relevant) LPS concentrations, and 
found that doses as low as 0.01 and 0.1 ng/ml can impair insulin-stimulated glucose transport (Fig. 8).    
 
   
Effect of TLR4 blockade on LPS- induced inflammation in human muscle cells.  Human myotubes were 
treated with 100 ng/ml LPS for 12 and 24 h in the presence/absence of the TLR4 inhibitor TAK242.  As shown 
in Figure 9, LPS caused a robust increase in the gene expression of IL-6 (9a) and MCP-1 (9b) in human muscle 
cells, an effect which was blocked by the TLR4 
inhibitor.  We have performed experiments evaluating 
the effect of TAK242 on insulin signaling.  Myotubes 
derived from a lean, nondiabetic subject were pre-
treated for 12 h with 100 ng/ml LPS in the 
presence/absence of 1µM TAK242, followed by 
stimulation with insulin.  As shown below, the TLR4 
inhibitor enhanced insulin-stimulated Akt (Fig. 10a) 
and AS160 (Fig. 10b) phosphorylation.    
 Sevelamer reduces LPS concentration and LPS-induced 
inflammatory responses.  Sevelamer is a non-absorbable, cross-linked 
polymer, which binds to LPS in the GI tract. Several studies have 
demonstrated sevelamer’s ability to reduce LPS plasma concentration in 
human subjects (35-37).  We tested the effect of sevelamer (1.6 g three 
times a day for 4 weeks; Genzyme, Cambridge, MA) on three T2DM subjects (mean age=65 years), and, 
consistent with the literature (35-37), sevelamer reduced plasma LPS level in all subjects (Figure 11).  
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Moreover, we evaluated the effect of sevelamer (ACIC, Coral Springs, FL) using our 
human myotube culture system, and found that pre-treatment with this agent decreased 
LPS concentration in the culture media (Fig. 12a), leading to a significant reduction 
in LPS-stimulated MCP-1 expression (Fig.12b).  Further, pretreatment with 
sevelamer also prevented LPS-induced activation of the IKK/NFκB pathway in 
isolated rat muscles (Fig. 12c).  
   
 
 
 
 
Sera from insulin resistant subjects causes an inflammatory response.  To examine whether the 
inflammatory state that occurs in muscle from insulin resistant subjects (38) is caused by a systemic factor, we 
incubated primary human myotubes derived from a lean person with sera from a lean and an obese person for 
24 h. The serum from the obese individual caused a significant inflammatory 
response as evidenced by increase MCP-1 (Fig. 13a) and IL-6 (Fig. 13b) 
gene expression. To examine whether LPS is the systemic factor inducing 
the inflammatory response, during the proposed study we will neutralize 
serum LPS with polymyxin B and will block TLR4 signaling in the 
human myotubes using TAK242 and short hairpin (sh)RNA.  
D) RESEARCH DESIGN AND METHODS 
Specific Aim 1) To determine the effect of high fat consumption on the intestinal microbiome, metabolic 
endotoxemia, and insulin action, in lean normal glucose tolerant subjects.  Rationale.  A high fat diet 
increases plasma LPS concentration in rodents (7, 26).  Moreover, a high fat ingestion also causes an elevation 
in LPS levels in human subjects (19, 30) and people who consume more fat have higher plasma LPS 
concentration (31).  In this Aim we will test whether a short-term high fat diet given to lean, normal glucose 
tolerant subjects increases intestinal permeability, plasma LPS level, and inflammation in the muscle.  Also we 
will test whether the inflammatory response and insulin resistance caused by high fat ingestion is prevented by 
interventions which reduce/prevent endotoxemia through different mechanisms:  
(i) A synbiotic (oligofructose plus Bifidobacterium longum): Probiotics are live bacteria which colonize the 
intestine after oral administration, prebiotics are products (nutritionals, etc.) that modulate the microbiota, and 
synbiotics are a combination of a probiotic and a prebiotic.  The prebiotic oligofructose is a nondigestible 
(resists hydrolysis by human digestive enzymes) fructooligosaccharide which is hydrolyzed and used as energy 
substrate by gut bacteria such as Bifidobacteria.  Bididobacteria are a dominant member of the intestinal 
microbiome and help to maintain integrity of the intestinal barrier (25, 39). Administration of oligofructose and 
the consequent increase in Bifidobacteria has been shown to protect the intestinal barrier, decrease intestinal 
permeability, lower plasma LPS concentration, and improve glucose metabolism in numerous studies conducted 
in animal models of insulin resistance (23-25).  In obese insulin resistant subjects with non-alcoholic 
steatohepatitis the administration of oligofructose and Bifidobacterium longum plus a lifestyle intervention 
caused a more significant decrease in plasma LPS concentration and improvement in insulin sensitivity 
(HOMA-IR) than lifestyle intervention alone (40). 
(ii) Sevelamer: This agent is a non-absorbable, cross-linked polymer that has strong LPS-binding capacity, and 
therefore limits intestinal wall translocation of LPS (41).  Sevelamer also has affinity for phosphates and it is 
FDA-approved for use as a phosphate binder in patients with chronic kidney disease. Sevelamer does not cause 
hypophosphatemia in individuals with normal kidney function (42, 43). We have confirmed sevelamer’s ability 
to reduce LPS concentration in vitro and to inhibit LPS-induced inflammatory response using our primary 
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human muscle cell culture system and isolated rat muscle preparation (Fig.12).  A cross-sectional study found 
that subjects who take sevelamer have lower LPS concentration (35) and prospective studies have shown that 
sevelamer reduces LPS concentration in humans as much as 80% (36, 37). We have confirmed sevelamer’s 
LPS-lowering effect in our study population (Fig. 11). Based upon its ability to sequester LPS in the intestinal 
lumen, sevelamer’s systemic anti-inflammatory and immune-modulating properties are being tested in a large 
NIH-sponsored multi-center (20 academic sites) trial, in otherwise healthy HIV-infected subjects with normal 
renal function (https://actgnetwork.org).   
Subjects.  36 (completers), healthy, lean (BMI <26 kg/m2) normal glucose 
tolerant subjects, aged 18-65 years, without a family history of T2DM, will be 
enrolled (50% males). Subjects will be randomized to receive a low or a high fat 
diet for 4 weeks.  After a 10-12 week washout period, subjects who first received 
the low fat diet, will then receive the high fat diet for 4 weeks, and vice versa 
(each subject will be its own control).  During the time the subjects receive these 
diets, they will simultaneously receive in a randomized double-blind fashion (i) 
placebo, (ii) synbiotic or (iii) sevelamer (n=12 completers per group). The    
protocol is outlined in Fig. 14. 
Inclusion Criteria: 1) Both genders.  All races and ethnic groups. 
2) Premenopausal women in the follicular phase, non-lactating, and with a negative pregnancy test.  
Postmenopausal women on stable dose of or not exposed to hormone replacement for ≥6 months.   
3) HCT≥ 34%, serum creatinine ≤ 1.4 mg/dl, electrolytes, urinalysis, and coagulation tests. LFTs up to 2X. 
4) Stable body weight (±2%) for ≥ 3 months. Two or less sessions of strenuous exercise/wk for last 6 months. 
Exclusion Criteria: 1) Presence of diabetes or impaired glucose tolerance based on ADA criteria. 
2) Current treatment with drugs known to affect glucose and lipid homeostasis. If the subject has been on a 
stable dose for the past 3 months, the following agents will be permitted: calcium channel blockers, β-
blockers, ACE inhibitors, angiotensin receptor blockers, and statins.  History of allergy to sevelamer. 
3) Non-steroidal anti-inflammatory drugs or systemic steroid use for more than a week within 3 months. 
4) Current treatment with anticoagulants (warfarin).  Aspirin (up to 325 mg) and clopidogrel will be permitted 
if these can be held for seven days prior to the biopsy in accordance with the primary physician. 
5) Use of agents that affect gut flora (e.g. antibiotics, colestyramine, lactulose, PEG) within 3 months. 
6) History of heart disease (New York Heart Classification greater than grade II; more than non-specific ST-T 
wave changes on the ECG), peripheral vascular disease, pulmonary disease, smokers. 
7) Poorly controlled blood pressure (systolic BP>170, diastolic BP>95 mmHg). 
8) Active inflammatory, autoimmune, hepatic, gastrointestinal, malignant, and psychiatric disease.  
9) History of gastrointestinal surgery or gastrointestinal obstruction within two years. 
Design.  Subjects will be recruited through the General Medicine Clinics of the Texas Diabetes Institute and the 
Audie L. Murphy VA Medical Center, and through local advertisement.  Study visits will be performed in the 
Bartter Research Unit (BRU) located in the VA Medical Center, which is funded by a Clinical and Translational 
Science Award (CTSA) to the University of Texas Health Sciences Center at San Antonio (UTHSCSA). 
Visit 1:  Subjects will come to the BRU at 7 AM for a medical history, physical examination (including 
anthropometric measurements), screening tests (CBC, chemistry, lipid profile, HbA1c, PT, PTT, UA), and ECG.   
Visit 2:  Within 3-7 days from Visit 1, eligible subjects will return at 7 AM after an overnight fast for an oral 
glucose tolerance test (OGTT).  A blood (LPS) and stool sample will be collected before the OGTT.  
Nutritional Intervention: After this visit subjects will begin a high or a low fat diet to be ingested for 28 days.  
The subjects will meet with a nutritionist (Celia Darland, RD) who will perform an assessment of macronutrient 
and energy intake.  The meals will be prepared in the Metabolic Kitchen of the BRU.  The nutritionist will 
design the nutritional components and provide dietary education.  The high and low fat diets will be designed 
based on the subjects’ usual dietary habits and energy intake to ensure palatability and adherence. The high fat 
diet will provide 60% of energy from fat (of which 50% from saturated fat), 15% of energy as CHO and 25% 
from protein. The low fat diet will provide 55% of energy from CHO, 20% from fat, and 25% from protein.  
6

 
The diets will be isocaloric in relation to the subjects’ habitual energy intake.  Ready-made meals will be 
provided to subjects. Oil, butter, heavy cream, eggs, mayonnaise and cheese will be used as the primary fat 
sources. Subjects will record food intake and will maintain their usual physical activity level. 
Pharmacological/nutraceutical intervention: On the same day that the nutritional intervention is started, 
subjects will be randomized to receive, in a double-blind fashion, placebo (maltodextrin, 6 g three times a day), 
the synbiotic [5 g of oligofrucose + 1 g Bifidobacterium longum (4x10^10 CFU/g) three times a day], or 
sevelamer (1.6 g sevelamer + 4.4 g maltodextrin three times a day), for 4 weeks, and discharged home.  
Sevelamer is obtained from Amgen (Renvela, Cambridge, MA), oligofructose from Orafti (Tienen, Belgium), 
Bifidobacterium from Danisco (Copenhagen, Denmark) and maltodextrin from American International Foods 
(Grand Rapids, MI).  Randomization and drug preparation/dispensing is performed by the Research Pharmacist 
of the BRU. For each dose the powder will be included in identical packets.  The subjects from the 3 groups 
(placebo, synbiotic, sevelamer) will ingest the powder contained in one packet with water 3 times /day for 28 
days. Before discharge, subjects are given printed instructions and information about potential side effects.    
Visit 3:  On day 3 of the nutritional intervention subjects will come to the BRU at 7 AM for evaluation of 
postprandial LPS concentration.  Subjects will ingest their breakfast according to their assigned meal plan.  
Plasma LPS, chylomicron LPS, insulin, and glucose levels are measured at times -30, +60, +120, and +180 min.   
Visit 4:  On day 24 of the intervention subjects will come to the BRU at 7 AM in the fasting state to undergo 
stool sample collection and an 8 h intestinal permeability (lactulose/mannitol) testing.     
Visit 5:  On day 28 subjects will undergo whole body DEXA, a euglycemic hyperinsulinemic clamp and indirect 
calorimetry as described (44, 45).  DEXA is performed to examine the effect of the interventions on body 
composition.  At time -30 min a biopsy of the vastus lateralis muscle will be performed.  At time 0 min, insulin 
will be infused at 60 mU/m2.min for 180 min.  A 2nd biopsy is performed on the contralateral leg at 180 min.   
After Visit 6, subjects will undergo a washout period of 10-12 weeks.  Subjects will not receive any treatment 
and will maintain their usual (isocaloric) alimentary habits and physical activity level.  They will meet every 
two to four weeks with the nutritionist to monitor their body weight.  
Visit 6:  After the 10-12 week washout period, subjects will come to the CRC for stool collection and plasma 
LPS measurement.  Subject will then begin the second nutritional intervention.  If subjects were placed on a low 
fat diet first, now they will begin a high fat diet, and vice versa.  During the second nutritional intervention 
subjects will receive the same treatment (synbiotic, sevelamer or placebo) assigned during the first intervention. 
Visit 7:  On day 3 of the second nutritional intervention subjects will undergo the same procedure as Visit 3 
(plasma LPS, insulin and glucose, and LPS content in chylomicrons, in the postprandial state).  
Visit 8:  On day 24 subjects come to the BRU for stool collection and intestinal permeability testing. 
Visit 9:  On day 28 of the second nutritional intervention subjects will come to the BRU at 7 AM to undergo 
DEXA, insulin clamp, 
indirect calorimetry, 
and biopsies of the 
muscle (2) as described 
above for Visit 5. 
Analyses.   Plasma 
LPS (LAL assay, 
Lonza, Walkersville, MD), LBP (Cell Sciences, Canton, MA), soluble CD14 (R&D Systems, Minneapolis, 
MN), IL-6 (R&D Systems), and TNFα (R&D Systems) concentration will be measured in all visits to examine 
the dynamics of LPS concentration and action (LPB, soluble CD14) during the course of the nutritional and 
pharmacologic/nutraceutical interventions.  Because turbidity from triglycerides can falsely increase LPS level 
(46), 0.25 vol of 50% acetic acid are added to each sample to eliminate turbidity (19).  In the basal muscle 
samples we will perform assays of TLR4 [IKK/NFκB and MAPK (JNK, p38, ERK)] signaling (75 mg tissue), 
TLR4 content (20 mg), inflammatory gene (TLR4, IL-6, MCP-1, iNOS, TNFα) expression (15 mg) and IRS-1 
ser phosphorylation (20 mg) as described by our group (38, 47, 48).  These measurements will establish whether 
elevations in plasma LPS caused by the high fat diet stimulate TLR4 and enhance IRS-1 ser phosphorylation 
7

 
which impairs insulin action.  On all muscle samples we will perform assays of insulin signaling (75 mg).  
These measurements will establish whether high fat diet-induced increases in LPS level and stimulation of 
TLR4 lead to impaired insulin-stimulated IRS-tyr phosphorylation, PI 3K activity, and Akt and AS160 
phosphorylation (49).  Stool samples will be collected for characterization of the intestinal microbiome through 
16S DNA sequencing.  This will allow us to evaluate the effect of the nutritional and 
pharmacologic/nutraceutical interventions on intestinal microbiota composition and how this relates to intestinal 
permeability, changes in LPS, inflammation (TLR4 signaling), and insulin action.   
Interpretation and limitations.  Based on prior animal studies we anticipate that that the high fat diet will 
modify the composition of the intestinal microbiota towards an unfavorable microbiome profile.  Specifically, 
we anticipate that there will be a reduction in Bifidobacteria content (22).  Since Bifidobacteria protect the 
integrity of the intestinal barrier (24, 39) we expect that the high fat diet will enhance intestinal permeability, 
which will lead to increased plasma LPS concentration.  If our hypothesis is correct, the elevation in LPS will 
trigger an inflammatory response in muscle, manifested by increased flux through TLR4 (TLR4-MyD88 
association, activation of IKK//NFκB and MAPK).  We predict that TLR4 stimulation will result in impaired 
insulin signaling (enhanced IRS-1 ser phosphorylation and decreased IRS-1 tyr phosphorylation, PI 3-kinase 
activity, and Akt-AS160 phosphorylation) and reduced peripheral insulin sensitivity (low Rd) during the clamp. 
Based on studies in rodents (24, 39), we predict that the synbiotic will ameliorate the damage to the intestinal 
epithelium (i.e. permeability) and the rise in plasma LPS caused by the high fat diet, which in turn will blunt the 
inflammatory response (TLR4 signaling) and improve insulin resistance.   
          In this Aim the effect of the LPS-binding agent sevelamer also will be assessed to provide an independent 
way for reducing plasma LPS concentration.  Based on previous human studies (35, 36) and our preliminary 
studies (Fig. 11) demonstrating that sevelamer decreases LPS concentration, and its capacity to bind LPS and 
inhibit its inflammatory effect (Fig. 12) (41), we anticipate that sevelamer-induced reductions in plasma LPS 
concentration will lead to reduced inflammatory signaling (IKK/NFκB, MAPK, inflammatory genes) in muscle. 
Because sevelamer’s effect is to reduce LPS, also we anticipate that flux through TLR4 (TLR4-MyD88 
association) will be lower.  In addition, we expect that the reductions in LPS concentration will predict 
(correlate with) changes (reductions) in TLR4 signaling and improved insulin signaling and sensitivity.   
          We anticipate that the microbiome changes and increases in plasma LPS will result in peripheral insulin 
resistance.  However, some (50, 51), but not all studies (52) have found reductions in insulin sensitivity in 
healthy subjects given short term isocaloric high fat diets.  The discrepant results likely are related to 
differences in nutritional interventions and populations studied.  We recognize that there may be variability in 
individual responses to the high fat diet.  In fact, we believe that is an advantage of the proposed experiments, 
which will allow us the test each subjects’ susceptibility to the nutritional intervention, and whether individual 
variations in microbial changes, correlate (predict) with changes in intestinal permeability, plasma LPS, and 
insulin sensitivity. These findings would lead to future studies designed to determine the mechanisms 
underlying such variability.   
         Insulin resistance is a multifactorial condition.  In addition to microbiome changes (tested in this Aim), 
other potential mechanism by which excess fat supply likely impairs insulin action include changes in free fatty 
concentration, toxic intracellular lipid metabolites, adipocyte- and macrophage-derived cytokines and 
mitochondrial function.  Because of these different mechanisms, and the interplay between them, we do not 
anticipate that targeting the microbiome and its products (LPS) solely will result in complete prevention of high 
fat diet-induced insulin resistance.  This proposal is based on the concepts that metabolic endotoxemia is an 
important contributing mechanism towards insulin resistance, and that targeting the microbiome and 
endotoxemia will ameliorate the deleterious effect of high fat consumption on inflammation and glucose 
metabolism.  While assessing the role of these different factors (lipid metabolites, mitochondrial function, etc.) 
is beyond the scope of the current application, the data and specimens collected during the study can be utilized 
in the future to examine the contribution of all these different mechanisms. 
         This Aim will examine whether high fat ingestion increases plasma LPS by altering intestinal permeability 
(Fig. 1).  However, another potential mechanism involves postprandial formation of chylomicrons which carry 
LPS (27).  An in-depth analysis of this mechanism is beyond the scope of this grant. However, samples will be 
8

 
collected in the postprandial state, and this will allow us to evaluate acute postprandial changes in chylomicron 
LPS content and plasma LPS level, and how these relate to measures of insulin action. 
Specific Aim 2) To determine whether microbiome modulation and an experimental reduction in plasma 
LPS concentration improve inflammation and insulin action in insulin resistant (obese and T2DM) 
subjects.  Rationale.  Obese and T2DM subjects have alterations in the intestinal microbiome (lower content of 
beneficial bacteria such as Bifidobacteria) (5), abnormal gut permeability (24, 53), increased plasma LPS 
(preliminary data) (54, 55), and elevated TLR4 signaling in muscle (38) and monocytes (56). In this Aim we 
will test whether protecting the intestinal barrier with a synbiotic will restore normal gut permeability, reduce 
LPS, and ameliorate inflammation (TLR4 signaling) and insulin resistance. To assess the role of LPS 
independently, also we will reduce plasma LPS concentration with sevelamer. 
Subjects.  36 (completers) lean (BMI<26 kg/m2) normal glucose tolerant, 36 (completers) obese (BMI=30-37) 
normal glucose tolerant, and 36 (completers) obese (BMI=30-37) T2DM subjects will be studied. 12 subjects 
per group will receive the symbiotic (oligofructose plus Bididobacterium longum), 12 sevelamer, and 12 
placebo. We will study an equal number of males and females.  Other inclusion and exclusion criteria are 
similar as described in Aim 1, except that T2DM subjects (HbA1c ≤ 8.5%) will be enrolled.  The only 
antidiabetic drugs allowed are sulfonylureas, due to their minimal effect on muscle physiology and 
inflammation. Sulfonylureas will be held for 48 h before any procedure to avoid hypoglycemia during fasting.   
Design. Visit 1:  Subjects will undergo a medical history, physical examination, and screening blood tests.  
Visit 2:  Within 3-14 days eligible subjects will return for OGTT. 
Visit 3:  Within 3-14 days subjects undergo blood (LPS) and stool collection and an intestinal permeability test. 
Visit 4:  Within 7-14 days from Visit 3, subjects will return to undergo a whole body DEXA, indirect 
calorimetry and an insulin clamp. Muscle biopsies will be performed before (-30 min) and at the end (+180 
min) of the clamp. Following completion of these studies, subjects will be randomized to receive in a double-
blind fashion, placebo (maltodextrin, 6 g three times a day), the synbiotic [5 g of oligofrucose + 1 g 
Bifidobacterium longum (4x10^10 CFU/g) three times a day], or sevelamer (1.6 g sevelamer + 4.4 g 
maltodextrin three times a day), for 4 weeks, and discharged to home.  While at home, subjects will maintain 
their habitual (isocaloric) diet and physical activity level.   
Visit 5: On day 24 of 
treatment blood (LPS) and stool are collected 
and 
an intestinal permeability test is done. 
Visit 6: On day 28, subjects undergo a 
second DEXA, indirect calorimetry, and 
insulin clamp with muscle biopsies.     
 
Analyses.  Plasma LPS, LBP, soluble CD14, IL-
6, TNFα and phosphate will be measured before, during, and after treatment (Visits 3, 4, 5 and 6).  Assays of 
TLR4 and insulin signaling in muscle are performed as described for Aim 1.  Stool samples will be collected 
before and during treatments.  This will allow us to evaluate differences in microbiome composition between 
lean, obese, and T2DM subjects at baseline, and establish the relationship between treatment-induced changes 
in specific bacteria composition/distribution with changes in inflammation and insulin action.   
Interpretation and limitations.   We expect that the obese and T2DM subject will have an unfavorable 
intestinal microbiome profile (decreases in Bifidobacteria and Bacteroidetes) (54, 55) which will be associated 
with enhanced intestinal permeability and increased LPS level.  Thus, we expect that these subjects will have an 
elevated inflammatory tone (TLR4 signaling) in muscle, which will reduce insulin action. Based upon previous 
animal studies (23-25), we anticipate that the synbiotic will restore gut epithelial integrity, decrease circulating 
LPS level, and ameliorate insulin resistance in obese and T2DM subjects. We also anticipate that sevelamer will 
decrease LPS concentration (36, 37) and will lead to reduced inflammatory signaling (IKK/NFκB, MAPK, 
inflammatory genes) in muscle.  Because sevelamer’s effect is to reduce LPS, also we anticipate that flux 
through TLR4 (TLR4-MyD88 association) will be lower.  In addition, we expect that the reductions in LPS 
concentration will predict (correlate with) changes (reductions) in TLR4 signaling and improved insulin action.  
9

 
          Aim 1 will test whether a fat-rich diet alters the gut microbiome and disrupts the intestinal barrier in lean 
subjects resulting in increased LPS translocation. The goal of Aim 2 is different; in this Aim we are proposing a 
proof-of-principle experiment to evaluate whether modulating the microbiome and lowering plasma LPS 
improves insulin action in obese and T2DM subjects. We anticipate that modulating the microbiome and plasma 
LPS concentration will decrease inflammation and insulin resistance in these individuals.  If these changes are 
the result of protection of gut barrier integrity from the increased fat intake which is common in obese and 
T2DM subjects, then sevelamer and the synbiotic also should prevent the inflammatory response and insulin 
resistance caused by the high fat diet in the lean subject from Aim 1. If the lean subjects are not protected from 
the high fat diet, but the obese and T2DM subjects benefit from sevelamer and the synbiotic, then 
improvements in inflammation and insulin action are likely caused by a mechanism different from protection 
against fat-induced damage. Other possible mechanisms by which LPS sequestration and/or microbiome 
modulation could improve inflammation and insulin action include changes in fructose-mediated endotoxemia 
(57), food energy extraction (9), appetite (10-13), bacterial flagellin levels (14), and peptidoglycans (15).   
          One limitation of the proposed experiment is that the synbiotic could alter inflammation and glucose 
metabolism through other mechanisms, such as by inducing changes in the concentration of FFA and cytokines.  
Sevelamer also could lead to other effects; this agent can bind to uric acid and reduce its plasma concentration 
(58), although uric acid per se generally is not thought to induce insulin resistance.  A primary human cell 
culture system will be employed in Aim 3 which will allow us to test whether the differences in inflammation 
and insulin sensitivity between groups and the effect of the interventions are due to changes in circulating LPS. 
         The proposed studies focus on the impact that the gut microbiome and endotoxemia have on inflammation 
and insulin action at the level of the skeletal muscle.  We are focusing on this tissue because it is the main site 
for insulin-mediated glucose disposal.  Nonetheless, the liver, adipose tissue, brain, and inflammatory cells 
(monocytes) also are potentially subjected to the influence of the microbiome and LPS.  Studying these 
organs/tissues is beyond the scope of this grant, but they are important areas for future investigation.   
Specific Aim 3) To determine whether the LPS present in the sera from lean high fat fed, obese, and 
T2DM subjects mediates the insulin resistance, and whether this effect is mediated via TLR4.  Rationale.  
An in vitro system will be employed because it will allow us to directly asses the role of LPS and TLR4 on 
insulin resistance in human muscle.  Testing the effect of sera in cultured cells while blocking the immune 
mediator of interest is a commonly employed method to assess the independent biological effects of the 
numerous immune mediators (antibodies, cytokines, etc.) which are altered in patients with immune-mediated 
diseases (lupus, rheumatoid arthritis) (59).  In this Aim we will apply the same concept in view that obesity and 
T2DM are considered inflammatory (and metabolic) diseases.   
Experimental model.  We will use primary human myotubes developed from the muscle samples obtained in 
the lean subjects enrolled during Aim 1, as described by our group (38).  To avoid experimental variation, 
myoblasts from all lean subjects will be pooled, aliquoted, and frozen. Prior to each experiment an aliquot will 
be thawed, cells plated, and differentiated into myotubes.  We will use cells from ealry (2-3) passages. 
     Experiment 1.  Does sera from lean high fat fed, obese, and T2DM induce inflammation and insulin 
resistance and are these effects ameliorated by in vivo treatment with the synbiotic and sevelamer? 
Human myotubes are treated with sera from (i) lean subjects studied in Aim 1 who ingested a low and high fat 
diet and received the synbiotic, sevelamer, and placebo; and (ii) lean, obese and T2DM subjects who received 
the synbiotic, sevelamer, and placebo (Aim 2).  Treatments are conducted in a time-dependent (24h and 48h) 
and dose-dependent (10%, 20% and 40% serum in MEM culture media) manner.  After these incubations, cells 
are collected for measurement of TLR4 signaling (TLR4-MyD88 association, IKK/NFκB, MAPK, 
inflammatory genes) (38, 48).  To assess the effect on insulin action, after treatment with the subjects’ serum, 
cells are stimulated with insulin for 20 min.  Cells are then collected in lysis buffer for insulin signaling studies 
(49) or subjected to a glucose transport assay (60).  Increasing concentrations of insulin (0, 1, 10 and 100 nM) 
will be employed to determine whether the sera affects insulin responsiveness (shifting of the curve). 
     Experiment 2.  Does elevated LPS in the circulation mediate the inflammatory state and insulin 
resistance observed in muscle from lean high fat fed, obese, and T2DM subjects?  Serum samples from 
these subjects will be treated with polymyxin B which sequesters LPS and specifically blocks its biological 
10

 
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Basal
LPS
TLR4 mRNA Expression  
Scrambled
TLR4-silencing
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Basal
LPS
TLR4 mRNA Expression  
Scrambled
TLR4-silencing
activity (61).  Polymyxin B cannot be administered to human subjects due to its nephrotoxicity.  However, 
because of its capacity to sequester LPS, ongoing multicenter clinical trials are being conducted assessing the 
effect of extracorporeal hemoperfusion with polymyxin B absorption columns in patients with sepsis (62).  
Assays of TLR4 signaling, inflammatory gene expression, insulin signaling, and glucose transport will be 
conducted in the presence and absence of polymyxin B (20 µg/ml). 
     Experiment 3.  Does TLR4 mediate the pro-inflammatory state and insulin resistance observed in lean 
high fat fed, obese, and T2DM subjects? Myotubes from lean subjects will be treated with sera from lean high 
fat fed, obese and T2DM subjects, and the same experiments described in Experiment 1 will be conducted 
(TLR4 signaling, inflammatory genes, insulin signaling, glucose transport). To test whether the effects from the 
sera are TLR4-mediated, TLR4 will be blocked/inhibited using two different approaches: 
a) TAK242: This agent (Takeda Pharmaceuticals) binds specifically to TLR4 and inhibits signaling by 
preventing its interaction with the adapter proteins TRAM and TIRAP (63).   
b) shRNA:  To knock down TLR4 we will use adenoviral-driven shRNA (Vector Biolabs, Philadelphia, PA) as 
described by our group (38).  As negative control we will use a scrambled non-mammalian 
sequence (Ad-scrambled).  We will perform a dose-response study to determine the minimal 
amount of virus that will achieve maximal knock down efficient.  Based on our own experience, 
we anticipate that this dose will be in the 1x102 to 1x108 PFU/ml range.  Fig. 17 shows images of 
typical human myotubes developed in our lab infected with adenovirus tagged with green fluorescent protein.  
Figs.18a and 18b show that our method is highly effective to knock down TLR4.  
 
 
Interpretation and limitations.   Based on our preliminary studies (Fig. 13),  
we anticipate that sera from lean high fat fed, obese, and T2DM will induce an 
inflammatory response and insulin resistance as evidence by elevated 
IKK/NFκB and MAPK signaling and inflammatory gene expression, decreased 
insulin signaling, and decreased glucose transport, as compared with myotubes treated with sera from lean 
subjects. If our hypothesis is correct, sera from high fat fed, obese and T2DM subjects, collected after treatment 
with the synbiotic and sevelamer, will induce a less severe inflammatory response and insulin resistance, and 
the changes in LPS level after in vivo treatment will predict the in vitro response.   
          One limitation of this approach is that numerous systemic factors could be responsible for the 
inflammatory response and insulin resistance caused by sera from high fat fed, obese and T2DM subjects.  If 
these effects are mediated by LPS, its neutralization with polymyxin B should ameliorate them.  In addition, if 
the effect is mediated through TLR4, the inflammatory response and insulin resistance caused by the sera will 
be reduced/prevented by blocking/downregulating TLR4 with TAK242 and shRNA.  
         TLR4 regulates numerous signaling cascades, including IKK/NFκB, JNK, ERK, p38 and SOCS. One or 
more branches downstream of TLR4 might be responsible for the negative effect of LPS on glucose 
metabolism. Dissecting the role of each of these branches in LPS-induced insulin resistance is beyond the scope 
of this grant application. Nonetheless, using our primary cell culture system and shRNA, in the future we could 
perform an in-depth analysis about the contribution of each of these signaling branches. 
Statistical analysis.   Experimental results will be expressed as means ± SE. Comparisons of means between all 
the groups will be done by ANOVA for repeated measures (67). Associations between measures of LPS 
concentration vs.TLR4 signaling and vs. insulin signaling/sensitivity, within a group, will be determined by 
Pearson’s correlation. For tests of correlation coefficients between groups we will use the Fisher’s Z 
transformation (64).  We will also determine the relationship between plasma LPS concentration vs. TLR4 
signaling and LPS vs. insulin signaling/sensitivity, by using multiple regression analysis.  Scatter plots will be 
done to look for outliers and to verify linearity.  Prof. Alex McMahan is an experienced biostatistician who will 
direct the statistical analysis of this study. Based on our preliminary results and data from the literature (36, 65) 
regarding the variability of these measurements, we calculated that an n=12 subjects/group is required to obtain 
power (1-β) of 0.80 with a 2-sided test at α= 0.05 significance level. Assuming a ~20% dropout rate, 15 
11

 
subjects/group would be sufficient to achieve the required group sizes.  The following data and power analysis 
(2-tailed) were obtained from an ongoing human study (PI: Musi).                                       
Variable 
Mean±SD Lean  N 
Mean±SD Obese 
n 
Power 
LPS Concentration  
0.049±0.055  
12 
0.119±0.014 
12 
0.99 
Insulin Sensitivity (M)  
11.3±2.8 
12 
8.1±2.4 
12 
0.91 
Ins Stim IRS1-P-Y  
4.0±1.1 
12 
0.9±0.6 
12 
0.99 
Variable 
Mean±SD Lean  N 
Mean±SD T2DM 
n 
Power 
LPS Concentration 
0.049±0.055 
12 
0.130±0.071 
12 
0.99 
Insulin Sensitivity (M) 
11.3±2.8 
12 
4.2±1.7 
12 
0.99 
Ins Stim IRS-1 P-Y 
4.0±1.1 
12 
1.5 ±1.0 
12 
0.99 
  For the following variables, data were obtained from published human studies (36, 40, 65).  
Variable 
Mean±SD Pre-Sevel  N 
Mean±SD Post-Sevel 
n 
Power 
LPS Concentration 
3.6±3.0 
12 
1.2±2.3 
12 
0.99 
CRP Concentration 
4.8±2.4 
12 
0.44±0.24 
12 
0.99 
Variable 
Mean±SD Placebo  
N 
Mean±SD  Synbiotic 
n 
Power 
LPS Concentration 
87±14 
12 
54±10 
12 
0.99 
We performed the following calculations (two-tailed) using data from our preliminary in vitro experiments: 
Variable 
Mean±SD Lean Serum 
n 
Mean±SD Obese Serum 
n 
Power 
MCP-1 mRNA 
1.18±0.18 
12 
2.3±0.6 
12 
0.88 
IL-6 mRNA 
0.6±0.06 
12 
1.2±0.06 
12 
0.99 
Other specific methods:  Intestinal permeability test. Five g lactulose and two g mannitol dissolved in water 
are ingested, hourly urine and plasma samples are collected for 8 hours, and lactulose and mannitol are analyzed 
by gas chromatography (66, 67).  Lactulose and mannitol permeate only in minimal amounts across the normal 
intestinal wall and are not metabolized by the body.  In disease state, the intestine becomes preferentially 
permeable to lactulose than mannitol. Dr. Fanti (Co-I) has previously conducted this test (68). 
Metagenomics.  The metagenomics experiments will be carried out by Dr. Qunfeng Dong (Co-I), as described 
by his group (69-71). DNA is extracted using a kit from MoBio (Carslbad, CA) and following the protocol 
established by the Human Microbiome Project Consortium  (72). PCR-amplified 16S rDNA is sequenced using 
the Illumina HiSeq2000 platform, following the sequencing and data processing protocol designed by Caporaso 
(73). Taxonomy is assigned to each high-quality read based on the best matching Greengenes database sequence 
(74). Operational Taxonomic Units are clustered by using the QIIME toolkit (75). For Aim 1, ANOVA and t-
test (and their non-parametric versions) and Chi-square/Fisher’s exact test are applied to detect taxa with 
significantly different relative abundance either within the same individuals (i.e., low fat vs. high fat) or 
between treatments (placebo, synbiotic, sevelamer). Shannon, UniFrac, and Bray-Curtis indices are compared to 
quantify the community-level variability among the samples within and between individuals. Multiple test 
corrections (e.g., Benjamini-Hochberg-Yekutieli) are applied to control for false discovery rate. The response 
variable (microbial composition) is analyzed with the explanatory variables (insulin sensitivity, LPS level, etc.) 
to evaluate for associations by Generalized linear models. Models also are used for analyzing the influence of 
explanatory variables on the relative abundance of significantly changing bacterial taxa (or) communities with 
treatments. For Aim 2, the data analysis and comparisons between groups (obese, T2DM, and lean) and 
treatments (placebo, synbiotic, sevelamer) are done similarly as in Aim 1. 
Summary/Timetable.   We are very excited about this proposal which will study the role that the microbiome 
and endotoxemia play a key role in the pathogenesis of insulin resistance. The studies proposed are innovative 
because the role of the micobiome on insulin resistance is unclear and relevant because they could lead to the 
development of novel therapies for T2DM.  This application describes a 3 year program: 
Year 1: Start Aim 1          Year 2: Complete Aim 1; Start Aims 2 and 3        Year 3: Complete Aims 2 and 3 
Risks: a)  Muscle biopsy: Local hematomas occur in <2% of subjects.  b)  Sevelamer: This drug is FDA 
approved for use as a phosphate binder.  Sevelamer does not cause hypophosphatemia in subjects with normal 
kidney function (71, 76).  As a precaution, we will only enroll subjects with normal phosphorus plasma 
concentration, which will be monitored during treatment.  Sevelamer is a very safe drug; its main side effects 
are gastrointestinal. Subjects with a history of gastrointestinal obstruction and/or surgery within 2 years will be 
excluded.  c) Oligofructose and Bididobacteria administration is very safe.  Rarely these can cause bloating. 
12

 
2. RELEVANT PUBLICATION ABSTRACTS: N/A 
 
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4. MANUSCRIPTS: N/A 
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