A Weight Neutral, High Protein, Moderate Carbohydrate Diet for Treatment of Type 2 Diabetes Mellitus
Phase: NA
Conditions: Type 2 Diabetes Mellitus
Sponsor: University of Minnesota

A Low Biologically Available Glucose and High Protein Diet for Treatment of Type 2 
Diabetes Mellitus 
Abbreviated Title: The LoBAG Diet and Type 2 Diabetes Mellitus 
Alternate Abbreviated Title: The Diet and Diabetes Study 
Alternate Title: A weight neutral, high protein, moderate carbohydrate diet for type 2 diabetes mellitus 
Alternate Title: A weight neutral, high protein, moderate carbohydrate diet for treatment of type 2 
diabetes mellitus 
 
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Protocol Version Date: 03/29/2021 
 

Project Summary 
Diet is a cornerstone of type 2 diabetes mellitus (DM2) treatment. Current standard of care diet 
recommendations focus on caloric restriction and weight loss, goals which can be difficult for patients 
to achieve. A moderate carbohydrate, high protein diet has been designed specifically for people with 
DM2, and has shown promising results in preliminary studies. This diet, called the Low Biologically 
Available Glucose (LoBAG) Diet, does not require caloric restriction or weight loss to produce 
beneficial effects. Preliminary data are exciting but studies were done only in a small subject 
population and in a carefully controlled research setting that was not typical of life in the real world. 
The purpose of this project is to expand upon the preliminary studies and offer the LoBAG diet to a 
free-living population of people with DM2. In addition, as part of this project, we propose a pilot study 
to assess the changes in the gut microbiome that occur with this diet in people with DM2. 
Subjects with DM2 will be enrolled in a randomized, parallel group trial of the LoBAG diet or a control 
diet over a 12 week intervention period. The subjects will be free-living and will do food preparation on 
their own. The main intervention will be instruction in either the LoBAG diet or the control diet. 
Measures of glycemic control will be collected before, during, and after the diet intervention. Stool 
samples will be used to collect preliminary data on gut microbiome changes. The diets will be 
isocaloric and subjects will not be asked to lose weight during the study. Our specific aims include: 1) 
to determine if the LoBAG diet improves hemoglobin A1c (HgbA1c) in a free-living population with 
DM2, 2) to investigate the effects of the LoBAG diet on fasting and postprandial glucose and insulin, 
fructosamine, and lipids, and 3) to determine the effects of the LoBAG diet on the composition of the 
gut microbiome. The hypothesis to be tested is that the LoBAG diet will produce a significant 
reduction in HgbA1c when compared to the control diet. The significance of this project will be to 
expand the generalizability of the LoBAG diet to a free-living population and to potentially improve 
dietary recommendations for the treatment and control of DM2.  
 
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1. Background 
1.1. The LoBAG diet impressively improves glycemic control for subjects with DM2 in a 
controlled research setting. There remains a crucial need to translate these findings to a free-
living population with DM2.  
Diet has the potential to be a readily accessible and powerful tool for treatment of DM2. The specific 
components of the ideal diet, however, have not been clearly defined. According to the American 
Diabetes Association (ADA), there is no one ideal diet for treatment of DM2, and people with DM2 
should focus on an eating pattern rich in nutrient dense foods in appropriate portion sizes (1). For 
those who are overweight or obese, caloric restriction and weight loss is recommended. 
Unfortunately, this goal is not usually achieved by patients.  
There is both common perception and plentiful evidence that dietary alteration improves glycemic 
control in DM2 through caloric restriction and weight loss. Yet, studies of a moderate carbohydrate, 
high protein diet demonstrated impressive improvement in glycemic markers in subjects with DM2 in 
the absence of caloric restriction or weight change (2-5). This diet, the Low Biologically Available 
Glucose (LoBAGx) diet (subscript indicates percent carbohydrate content in diet), was designed by 
Nuttall and Gannon for treatment of DM2 (6). The rationale for the design came from two observations 
made in their previous work: glucose in the diet is the major determinant of postprandial plasma 
glucose levels, and dietary protein stimulates insulin secretion (6). In a randomized, crossover trial of 
the LoBAG20 diet (20% carbohydrate, 30% protein, 50% fat) compared to a control diet (55% 
carbohydrate, 15% protein, 30% fat) in 8 men with untreated DM2, glycated hemoglobin decreased 
from 9.8 ± 0.5% to 7.6 ± 0.3% (p < 0.001) over 5 weeks. In contrast, there was not a significant 
change in glycated hemoglobin with the control diet. In addition, both fasting and postprandial plasma 
glucose decreased with the LoBAG20 diet, as did the net mean 24 hour integrated plasma glucose 
area response, which decreased by 77%. Subject weight remained stable on both the LoBAG and 
control diets (3).  
  
Although the LoBAG20 diet was effective, the significant limitation of carbohydrate content compared 
to a typical American diet was felt to reduce acceptability. Consequently, the LoBAG30 diet (30% 
carbohydrate, 30% protein, and 40% fat) was designed with a goal of increasing acceptability of the 
diet for a general population of patients with DM2. The beneficial effect on glycated hemoglobin 
persisted. In a randomized, crossover trial of the LoBAG30 diet in 8 men with untreated DM2 (2 
completed only the test diet period) total glycated hemoglobin decreased from 10.8 ± 0.4% to 9.1 ± 
0.5% (p < 0.001) after 5 weeks of the LoBAG30 diet. Weight remained stable. There was a decrease in 
both fasting and postprandial plasma glucose concentrations with the LoBAG30 diet, and a decrease in 
24 hour plasma glucose area response (4). A second study of the LoBAG30 diet in 8 men with 
untreated DM2 demonstrated that the decrease in glycated hemoglobin continued after 5 weeks, with 
a decrease in mean glycated hemoglobin from 10.0% at baseline to 8.7% at 5 weeks and 7.5% at 10 
weeks (5). A main conclusion from the LoBAG30 studies was that the diet was similarly effective to the 
LoBAG20 diet in improving glycemic control in subjects with DM2, and contained a carbohydrate 
content that would be more acceptable to a broad range of patients. 
Although these are exciting data, their main limitation was the delivery of the diet in a controlled 
research setting in a small, all male population. Thus, it remains unknown and is clinically important to 
determine whether these promising results can be replicated in a free-living population of men and 
women with DM2. 
1.2. The gut microbiome may have an important role in the effectiveness of diet therapy for 
DM2, but its role is not yet understood.  
 
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Diet has important effects on the gut microbiome in mice and in healthy human subjects (7-11). It is 
increasingly recognized that the gut microbiome has a significant role in glucose metabolism (12), and 
that dysbiosis can lead to perturbations in normal metabolism (13; 14). It has been hypothesized that 
changes in bacterial metabolites, especially short chain fatty acids and secondary bile acids, is a 
mechanism by which the microbiome has an effect on metabolism (15-18). In human subjects with 
DM2, however, there is a knowledge gap. It remains unknown if the dietary recommendations given to 
patients with DM2 have an effect on the gut microbiome, and if so whether these changes confer 
metabolic benefit. Since the LoBAG diet alters macronutrient composition of the diet it would be 
expected to cause changes in the gut microbiome and bacterial metabolites. The importance of this is 
unknown.  
1.3. Significance  
The proposed study is significant because it will test the generalizability of the effects of the LoBAG 
diet in a general population of people with DM2. This could have important implications for DM2 
treatment. The study is innovative in its use of a weight neutral, isocaloric, high protein and moderate 
carbohydrate diet in a free-living population of subjects. There are 29 million people with DM2 in the 
US (19), and so expanding and improving treatments to maintain glycemic control is an important 
public health goal. In addition, no study to our knowledge has investigated the gut microbiome before 
and after a dietary intervention for DM2. By contributing to the limited current knowledge about gut 
microbiome, diet, and diabetes, the study will provide pilot data for future studies. The study will hope 
to reshape effective dietary recommendations for the treatment of DM2.  
2. Hypotheses 
We hypothesize that the LoBAG30 diet will produce a greater reduction in HgbA1c over 12 weeks in a 
free-living population of subjects with DM2 than a control diet consistent with ADA guidelines, without 
the need for weight loss. We have an additional exploratory hypothesis that the LoBAG30 diet will 
induce potential beneficial changes in the gut microbiome, including increased species diversity, 
increased relative abundance of species associated with a healthy microbiome profile, and increases 
in short chain fatty acids and secondary bile acids.  
3. Specific Aims to Test Hypotheses  
3.1. Specific Aim #1: To compare the reduction in HgbA1c over 12 weeks in a free-living 
population of subjects with DM2 who consume either the LoBAG30 diet or a control diet 
consistent with ADA guidelines.  
The LoBAG30 diet was chosen for study because of its high level of acceptability to subjects (5) and 
the likelihood that it could be used by a broad range of patients with DM2. The current ADA diet 
recommendations were used to develop the control arm of the trial because they represent the current 
standard of care in diabetes nutrition therapy. We hypothesize that, when compared to a control diet 
consistent with ADA guidelines, the LoBAG30 diet will produce a better HgbA1c outcome. 
3.2. Specific Aim #2: To investigate the effects of the LoBAG30 diet compared to the control diet 
on fasting and postprandial glucose and insulin, fructosamine, and lipids in a free-living 
population of subjects with DM2.  
Secondary endpoints to be assessed will include fasting plasma glucose, fasting serum insulin, 
postprandial glucose response, postprandial insulin response, fructosamine, and fasting serum lipids. 
It will be important to determine if the LoBAG diet reduces fasting and postprandial plasma glucose 
and affects serum insulin concentrations in this population. We also want to determine the effect of the 
LoBAG diet on fructosamine, which is a measure of glycemic control that reaches equilibrium more 
rapidly than does HgbA1c. Finally, we wish to be sure that the LoBAG diet does not have deleterious 
effects on the fasting lipid profile.  
 
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3.3. Specific Aim #3: To determine the effects of the LoBAG30 diet compared to the control diet 
on the composition of the gut microbiome.  
Stool samples collected before and after diet intervention will be used for species analysis of the gut 
microbiome. We hypothesize that there will be a change in the gut microbiome following the LoBAG30 
diet when compared to the control diet, and that this change will be associated with characteristics 
expected to provide metabolic benefit to subjects with DM2. These changes might include increased 
species diversity and increased relative abundance of species associated with a healthy microbiome 
profile. If funding can be secured, we will also analyze bacterial metabolites to determine if there are 
potentially beneficial increases in short chain fatty acids and secondary bile acids with the LoBAG30 
diet.    
4. Research Plan 
An overview of the proposed study design is shown in Figure 1. A detailed study timeline is included 
as an appendix to this document.  
Figure 1. Overview of proposed study design.  
 
 
 
 
 
4.1. Study cohort  
Up to 70 men and women with DM2 will be recruited and enrolled (dependent on funding), for a goal 
of 44 participants completing the trial.  
To be eligible, subjects must be >18 years of age, have a previous diagnosis of DM2 by ADA criteria 
(20) and have a HgbA1c of 7.0-9.5% while either taking no medications for diabetes or using 
metformin alone.   
Exclusion criteria will be: 
Week
1
2
3
4
5
6
7
8
 9
10
11
12
 
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Randomization
Visit 6 
Outcome 
measures 
(end of week 12) 
Visit 1 
Consent 
LoBAG30 Diet
ADA Compliant Control Diet 
Visit 2 
Baseline 
measures; 
Start diet 
Visit 4 
Midpoint 
measures; 
Reinforce 
diet advice 
Visit 3 
Reinforce 
diet advice 
Visit 5 
Reinforce 
diet advice 

1) type 1 diabetes mellitus,  
2) treatment with insulin,  
3) BMI <25 kg/m2,  
4) lack of weight stability defined as a change in weight of more than 5 pounds in the prior 3 months,  
5) estimated glomerular filtration rate < 60 ml/minute/1.73 m2, 
6) urine albumin >300 mg/g creatinine,  
7) pregnancy or immediate plans to become pregnant,  
8) breast feeding,  
9) dietary restriction(s) that would preclude consumption of study diets,  
10) inability or unwillingness to prepare meals,  
11) anemia, which would affect interpretation of HgbA1c,  
12) presence of any disease which would make adherence to the study protocol difficult, and  
13) use of antibiotics in the 3-month period prior to enrollment.   
Subjects will be recruited from the University of Minnesota and surrounding communities. In addition, 
potential subjects will be identified from the Fairview patient population using Informatics Consulting 
Services (ICS) through the CTSI, and mailed letters informing them of possible study eligibility by the 
Fairview Research Administration.  
4.2. Outcome Measures 
The primary endpoint will be HgbA1c at the end of the 12 week diet intervention period. Secondary 
endpoints will be:  
1) Weight 
2) Fasting plasma glucose 
3) Fasting serum insulin 
4) Postprandial glucose response at +30, +60, +90, +120, +180 and +240 minutes following the 
start of a meal consistent with the assigned diet 
5) Postprandial insulin response at +30, +60, +90, +120, +180 and +240 minutes following the 
start of a meal consistent with the assigned diet 
6) Fructosamine  
7) Fasting serum lipids 
8) Stool samples for microbiome species analysis. Samples will also be saved for possible 
bacterial metabolite analysis.  
9) Glucose variability as measured by continuous glucose monitoring (CGM). 
4.3. Intervention 
A randomized, controlled, parallel group design will be used to compare the effects of the LoBAG30 
diet or a balanced diet consistent with current ADA guidelines on our outcomes. Dietary intervention 
periods will be 12 weeks long. Both the LoBAG and control diets will consist of common foods.  
Subjects will be asked to keep their physical activity level constant during the study. Medications for 
diabetes will be continued without change during the study. Medications will be monitored, and if a 
participant’s medications are altered this will be noted. If antibiotics are used during the study this will 
be recorded. Subjects will be instructed that they do not need to monitor blood sugars regularly at 
home.  
After enrollment and consent subjects will be randomized in a 1:1 allocation ratio to the LoBAG30 diet 
or a balanced diet consistent with ADA guidelines. Randomization will be stratified by diabetes 
treatment at enrollment (no glycemic medications or metformin) and by HgbA1c (7.0-8.5% or 
8.5-9.5%), and implemented within stratum using randomly permuted block sizes of 2 and 4. If closely 
related subjects choose to enroll together (for example spouses or subjects that live together, and 
would typically share meals) they will be randomized to the same study arm. Baseline measures will 
be collected. Subjects will meet individually with the study dietitian, who will provide diet instruction, 
sample menus, and recipes consistent with the assigned diet. Dietitian consultation may be conducted 
 
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in person or by phone. During week 2 subjects will have a repeat consult with the dietitian to reinforce 
diet instruction. If weight has changed caloric intake will be adjusted as necessary for a goal of weight 
stability. A midpoint visit will be conducted at week 6, during which subjects will provide a blood 
sample, urine sample, and a stool sample, and will consult with the dietitian for reinforcement of diet 
advice. If weight has changed caloric intake will be adjusted to prevent further change. Midpoint blood 
and stool samples will be stored and analyzed in the future based on available funding. At week 9 
subjects will consult with the study dietitian for reinforcement of diet advice and collection of a urine 
sample to assess compliance. The final study visit will be at the end of week 12. All outcome 
measures will be assessed at the final visit. 
If funding is available continuous glucose monitoring (CGM) will be added at the beginning and end of 
the study intervention. Subjects will be asked to wear the Freestyle Libre Pro CGM system for 2 
weeks following the screening visit (visit 1) and for 2 weeks between visit 5 and visit 6. The CGM 
sensor will be worn on the back of the arm and will monitor and record interstitial glucose continuously 
throughout periods of CGM use. Subjects will be blinded to CGM glucose data. Sensors will be 
returned to study staff by mail or in person at study visits. Subjects will be given the option to receive 
a copy of CGM data, and to review this data with study staff, at the final study visit.  
4.4. Assessment of Compliance and Acceptability  
At baseline, week 2, week 6, week 9 and week 12 urine samples will be collected for urine nitrogen 
and creatinine. The ratio of nitrogen/creatinine will be used as a marker of compliance with the high 
protein LoBAG30 diet, as previously described (4). Subjects in both groups will complete surveys at 
week 1, week 6, and week 12 with questions about compliance, acceptability of the diets, and quality 
of life. A study investigator will contact subjects every two weeks during the diet intervention period to 
encourage diet compliance. Subjects will be asked to complete 3-day food diaries at the beginning, 
midpoint, and end of the 12 week study. If funding is available, an unannounced 24-hour diet recall 
will be conducted via telephone by the Nutrition Coordinating Center at the University of Minnesota. 
The recall will be administered by a trained interviewer and analyzed using Nutrition Data System for 
Research software. 
4.5. Subject Compensation 
Subjects will be provided a total stipend of $600 for completion of the study. This amount includes a 
weekly stipend to partially account for the increased price of required foods ($15 per week), 
compensation for time (average $15 per week), and a bonus for study completion ($240). Payments 
will be made at regular intervals during the study. If a subject withdraws from the study before 
completion, compensation will be provided for each week of participation up to the time of withdrawal. 
In addition to the above compensation, parking will be paid for at study visits.  
Subjects who come for a screening visit only (do not qualify to go forward with the study) will be 
mailed a check with compensation ($30). Subjects who enroll in the study will be given the option of 
payment using pre-paid debit cards with the Greenphire ClinCard system. If a subject chooses this 
option, they will be given a handout with instruction on use of the card. If a subject prefers not to use 
this option, they will be paid through checks mailed following study visits. The payment schedule will 
be the same with either reimbursement option, except that check processing may delay 
reimbursement by a few weeks.   
4.6 Data Collection  
Data will be collected by study investigators in source documents and will be entered into a RedCap 
database. Data will be identified using indirect identifiers. Hard copies of source documents will be 
stored in a locked office. All computers used for this study will be password protected. In addition the 
 
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RedCap database will be password protected and will only be accessible to those study investigators 
and staff who require access.  
Blood, urine, and stool specimens will be collected by the study investigators or Clinical and 
Translational Science Institute (CTSI) or Clinical Research Unit (CRU) staff. All specimens will be 
identified using indirect identifiers and appropriate numbering and labeling for date, visit number, and 
time of collection. Specimens will be either transported directly to the lab for processing or will be 
stored in CTSI freezers.  
The study PI will manage the study data and specimens, with assistance from the study coordinator. 
Records and frozen specimens will be stored indefinitely.    
Protected health information will not be saved for potiential participants who are screened but do not 
come in for a study visit and thus do not give signed informed consent. At the conclusion of the 
screening survey potential participants will be asked if they are willing to be contacted about future 
studies for which they may be eligible. If yes, their name, age at time of screen, gender, reason for 
study ineligibility, method of finding out about the study (flier, letter, etc), and contact information will 
be saved in a password protected log. All other protected health information collected during the 
screening survey will be destroyed.  
Following study enrollment all study participants will have a M Health medical record number (MRN) 
identified or created. The electronic health record (EPIC) may be accessed and used for scheduling 
purposes, as required by the clinical research unit (CRU).   
4.7. Methods of Data Analysis  
The primary outcome will be HgbA1c at the end of the 12-week study period. Results will be 
compared pre and post diet intervention (within an individual subject) using paired t-tests and between 
dietary interventions (LoBAG30 versus control) using unpaired t-tests. Data will be analyzed at study 
completion.   
Sample size was estimated a priori based on a two-tailed type I error of 0.05. For a difference of 1.5 
points in HgbA1c and a power of 80%, 12 participants are required per intervention group. This 
number was increased by 30% to account for participant loss during screening, and an additional 30% 
to account for participant attrition, yielding 19 participants per intervention group (total 38 participants). 
An additional 12 participants have been enrolled using pilot funding from the University of Minnesota 
(of which 8 were eligible and 6 completed the entire study intervention).   
This estimate uses preliminary data from existing LoBAG30 studies which were collected in a carefully 
controlled research setting. This differs from the conditions planned for this study. There are no data 
on the performance of the LoBAG30 diet in a free-living population of subjects with DM2. Thus, we will 
generate data that can be used to estimate effect size and variability for a power calculation for a 
future larger clinical trial. 
With a goal of 38 plus 6 subjects completing the entire 12-week intervention, we anticipate recruiting 
up to 70 subjects in total.  
If a subject withdraws from the study prior to study completion they will be invited to complete the final 
study visit prior to withdrawal, if possible. Any data collected from the subject prior to study withdrawal 
will be included in the final analysis. In the case of subject withdrawal an additional subject will be 
recruited to replace accordingly, dependent on funding.  
4.8. Data and Safety Monitoring Plan 
 
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Protocol Version Date: 03/29/2021 
 

University of Minnesota Institutional Review Board (IRB) approval will be obtained prior to study 
initiation. Data and safety monitoring will be conducted by the IRB, the PI, and independently by 
qualified staff appointed by the CTSI. Monitoring by the CTSI will occur annually at a minimum. The PI 
will have primary responsibility for recording of events, follow up, and reporting to the IRB. 
Any adverse events will be recorded immediately in the source document and in the appropriate study 
log for reporting to the IRB. The clinical course of any adverse event will be followed until resolution, 
stabilization, or until it has been determined that study participation is not the cause. Reports of all 
adverse events (including follow-up information) will be submitted to the IRB according to IRB 
requirements. Reports of any serious adverse events (defined as any event that is: 1) fatal 2) life 
threatening 3) requires or prolongs a hospital stay 4) results in persistent or significant disability or 
incapacity 5) results in a congenital anomaly or birth defect or 6) results in an important medical 
event) will be submitted to the IRB according to IRB requirements. Reports of any unanticipated 
problems involving risk to subjects or others (UPIRTSO) will be submitted to the IRB according to IRB 
requirements.  
Subjects may discontinue participation in the study at any time, for any reason. Subjects will be 
withdrawn from the study in the case of a significant adverse event, consent withdrawal, or failure of 
the subject to adhere to protocol requirements. Failure to adhere to the protocol will be defined as 
compliance with <80% of requested study activities. In addition, any subject observed to have 
unacceptable responses to research procedures or to be unable to safely tolerate participation in the 
study as judged by the study investigator will be withdrawn.  
The study will be stopped if >50% of subjects cannot meet compliance with >80% of study activities. 
This rule will be implemented after enrollment of the first 6 subjects.  
4.9. Anticipated Problems and Proposed Solutions  
Although a free-living study cohort was purposefully chosen to answer the central question of this 
study, it is anticipated that compliance with study diets in this cohort will be lower than that in previous 
LoBAG diet studies. To address this, measures of compliance have been included, and frequent 
phone calls added to the protocol to encourage diet compliance. Although the dietary interventions in 
this study are meant to be weight stable, we acknowledge that weight change during the study may 
influence our results. For this reason, we have included three predetermined points in the protocol 
when caloric advice can be adjusted if indicated. Finally, use of medications for diabetes could impact 
the size of the effect of the LoBAG30 diet on HgbA1c, and so we have limited inclusion criteria to no 
glycemic medication or metformin only. We will also use stratified randomization to ensure equal 
distribution of medication use across study groups.  
5.0 Retention of Study Records 
Study records and data will be retained following comple6on of the study, in compliance with NIH and 
University of Minnesota requirements. Signed and dated HIPAA authoriza6ons and consent documents will be 
maintained for at least 6 years aEer comple6on of the research. Other study records will be maintained for at 
least 3 years aEer comple6on of the research.  
 
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References  
1. American Diabetes Association: Foundations of care: education, nutrition, physical activity, smoking 
cessation, psychosocial care, and immunization. Diabetes care 2015;38:S20–S30 
2. Gannon MC, Nuttall FQ, Saeed A, Jordan K, Hoover H: An increase in dietary protein improves the blood 
glucose response in persons with type 2 diabetes. American Journal of Clinical Nutrition 2003;78:734-741 
3. Gannon MC, Nuttall FQ: Effect of a high-protein, low-carbohydrate diet on blood glucose control in people 
with type 2 diabetes. Diabetes 2004;53:2375-2382 
4. Nuttall FQ, Schweim K, Hoover H, Gannon MC: Effect of the LoBAG(30) diet on blood glucose control in 
people with type 2 diabetes. British Journal of Nutrition 2008;99:511-519 
5. Gannon MC, Hoover H, Nuttall FQ: Further decrease in glycated hemoglobin following ingestion of a 
LoBAG(30) diet for 10 weeks compared to 5 weeks in people with untreated type 2 diabetes. Nutrition & 
Metabolism 2010;7 
6. Gannon MC, Nuttall FQ: Control of blood glucose in type 2 diabetes without weight loss by modification of diet 
composition. Nutrition & Metabolism 2006;3 
7. Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI: The effect of diet on the human gut 
microbiome: a metagenomic analysis in humanized gnotobiotic mice. Science translational medicine 
2009;1:6ra14 
8. Hildebrandt MA, Hoffmann C, Sherrill-Mix SA, Keilbaugh SA, Hamady M, Chen YY, Knight R, Ahima RS, 
Bushman F, Wu GD: High-fat diet determines the composition of the murine gut microbiome independently of 
obesity. Gastroenterology 2009;137:1716-1724 e1711-1712 
9. Murphy EF, Cotter PD, Healy S, Marques TM, O'Sullivan O, Fouhy F, Clarke SF, O'Toole PW, Quigley EM, 
Stanton C, Ross PR, O'Doherty RM, Shanahan F: Composition and energy harvesting capacity of the gut 
microbiota: relationship to diet, obesity and time in mouse models. Gut 2010;59:1635-1642 
10. Wu GD, Chen J, Hoffmann C, Bittinger K, Chen YY, Keilbaugh SA, Bewtra M, Knights D, Walters WA, Knight 
R, Sinha R, Gilroy E, Gupta K, Baldassano R, Nessel L, Li H, Bushman FD, Lewis JD: Linking long-term dietary 
patterns with gut microbial enterotypes. Science (New York, NY) 2011;334:105-108 
11. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, Ling AV, Devlin AS, Varma Y, 
Fischbach MA, Biddinger SB, Dutton RJ, Turnbaugh PJ: Diet rapidly and reproducibly alters the human gut 
microbiome. Nature 2014;505:559-563 
12. Cani PD: Metabolism in 2013: The gut microbiota manages host metabolism. Nature reviews Endocrinology 
2014;10:74-76 
13. Tilg H, Moschen AR: Microbiota and diabetes: an evolving relationship. Gut 2014;63:1513-1521 
14. Caricilli AM, Saad MJA: Gut microbiota composition and its effects on obesity and insulin resistance. Current 
Opinion in Clinical Nutrition and Metabolic Care 2014;17:312-318 
15. Puddu A, Sanguineti R, Montecucco F, Viviani GL: Evidence for the gut microbiota short-chain fatty acids as 
key pathophysiological molecules improving diabetes. Mediators of inflammation 2014;2014:162021 
16. den Besten G, Bleeker A, Gerding A, van Eunen K, Havinga R, van Dijk TH, Oosterveer MH, Jonker JW, 
Groen AK, Reijngoud DJ, Bakker BM: Short-Chain Fatty Acids protect against High-Fat Diet-Induced Obesity via 
a PPARgamma-dependent switch from lipogenesis to fat oxidation. Diabetes 2015; 
17. Hartstra AV, Bouter KE, Backhed F, Nieuwdorp M: Insights into the role of the microbiome in obesity and 
type 2 diabetes. Diabetes care 2015;38:159-165 
18. van Olden C, Groen AK, Nieuwdorp M: Role of Intestinal Microbiome in Lipid and Glucose Metabolism in 
Diabetes Mellitus. Clinical Therapeutics 2015;37:1172-1177 
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Its Burden in the United States, 2014. Department of Health and Human Services 2014; 
20. American Diabetes Association: Classification and diagnosis of diabetes. Diabetes care 2015;38 Suppl:S8-
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Appendix 1: Timeline and Details of Study Visits 
 
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Screen. No visit needed. Will usually be done by telephone; may also be done by email or in person. 
(20 minutes)  
Visit 1. Consent and Screening Visit. (1 hour) 
-
Review consent document and obtain informed consent.  
-
Collect baseline compliance survey, Diet Satisfaction Questionnaire, and Diabetes Treatment 
Satisfaction Questionnaire. Can alternatively be collected at visit 2.   
-
Measure weight and height.  
-
Non-fasting blood draw (for HgbA1c, basic metabolic panel, and complete blood count) 
-
Collect urine sample (for baseline urine nitrogen and urine creatinine, urine microalbumin, and 
pregnancy test if female and premenopausal).  
-
Send home with 3-day food intake diary (to be used by the study dietitian for caloric planning; 
the same food diary will also be collected at visit 4 and visit 6).  
-
If funding allows, place Abbott Freestyle Libre CGM. Provide supplies to return the CGM 
sensor (by mail or in person). 
-
Provide stool collection kit. Give instruction in stool sample collection.  
Details for stool sample collection: 
-
Subject self-collects at home by sampling a stool with a sterile swab and putting the 
sample in a collection tube containing a preservative.  
-
This room-temperature-stable sample can be delivered to study investigators by the 
subject at study visits, or can be returned through the mail.  
-
Study staff will put the de-identified sample into the CTSI storage freezer.  
Randomization. No visit needed.  
NCC Diet Recall 
-
If funding allows, telephone call conducted by the Nutrition Coordinating Center (NCC) for an 
unannounced 24 hour diet recall (30 minutes; prior to visit 2/diet start).  
Visit 2. Diet Instruction and Baseline Labs. (5 hours; beginning of Week 1) 
-
Subjects bring in stool sample or return by mail.  
-
If applicable, subjects bring in CGM sensor or return by mail.  
-
Collect weight, height, blood pressure. 
-
Fasting blood draw (for plasma glucose, serum insulin, lipid panel, fructosamine, and a stored 
sample). 
-
Provide a test meal consistent with the assigned diet (Control diet arm: 50% carbohydrate, 
15% protein, 35% fat or LoBAG30 diet arm: 30% carbohydrate, 30% protein, 40% fat). The 
composition of the control diet was chosen to be consistent with typical macronutrient intake in 
the United States (21) and consistent with ADA guidelines (1). In addition to the fasting 
samples, obtain plasma glucose and serum insulin at +30, +60, +90, +120, +180 and +240 
minutes following the start of the meal. 
-
Collect 3-day food diary.  
-
Collect list of current medications.  
-
Collect baseline compliance survey, Diet Satisfaction Questionnaire, and Diabetes Treatment 
Satisfaction Questionnaire if not already administered at Visit 1.  
-
Meeting with study dietitian for diet instruction. Sample menus and recipes consistent with the 
assigned diet will be provided. Dietitian consult may be conducted by phone if needed to 
facilitate scheduling.  
-
If funding allows, provide Actigraph Activity Monitor for subject to wear between Visits 2 and 
Visit 3. 
Visit 3. Diet Reinforcement. (1 hour; Week 2)  
-
If applicable, subjects return Actigraph Activity Monitor.  
-
Measure weight.  
 
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-
Collect urine sample for urine nitrogen and urine creatinine.  
-
Meeting with study dietitian for reinforcement of diet advice. Adjust caloric intake if needed for 
goal of weight stability. Dietitian consult may be conducted by phone. If dietitian consult is 
conducted by phone the participant will be encouraged, but not required, to come to the study 
center for weight and urine sample collection (this exception applies to visit 3 only).  
Visit 3.1. (5-10 minutes; Week 4) 
-
Telephone call conducted by study investigator or coordinator to encourage study 
compliance. 
  
Visit 4. Midpoint Visit. (1 hour; Week 6)  
-
Subjects bring in stool sample or return by mail.  
-
Measure weight and blood pressure.  
-
Fasting or non-fasting blood draw for HgbA1c, fructosamine, and a stored sample.  
-
Collect urine sample for urine nitrogen and urine creatinine.  
-
Meeting with study dietitian for reinforcement of diet advice. Adjust caloric intake if needed for 
goal of weight stability. Dietitian consult may be conducted by phone. 
-
Collect 3-day food diary.  
-
Collect list of current medications. 
-
Collect compliance survey and Diabetes Treatment Satisfaction Questionnaire 
Visit 4.1. (5-10 minutes; Week 8)  
 
-
Telephone call conducted by study investigator or coordinator to encourage study 
compliance. 
Visit 5. Diet Reinforcement. (1 hour, week 9-10) 
-
Measure weight.  
-
Collect urine sample for urine nitrogen and urine creatinine.  
-
Meeting with study dietitian for reinforcement of diet advice. Adjust caloric intake if needed for 
goal of weight stability. Dietitian consult may be conducted by phone.  
-
If funding allows, place Abbott Freestyle Libre CGM. Provide supplies to return the CGM 
sensor (by mail or in person). 
-
If funding allows, provide Actigraph Activity Monitor for subject to wear between Visits 5 and 
Visit 6. 
Visit 5.1. (5-10 minutes; Week 11)  
 
-
Telephone call conducted by study investigator or coordinator to encourage study 
compliance. 
NCC Diet Recall 
-
If funding allows, telephone call conducted by the Nutrition Coordinating Center (NCC) for an 
unannounced 24 hour diet recall (30 minutes; Week 9-12).  
Visit 6. Final Visit (5 hours; end of Week 12)  
-
Subjects bring in stool sample or return by mail.   
-
If applicable, subjects return Actigraph Activity Monitor.  
-
If applicable, subjects bring in CGM sensor or return by mail.  
-
Collect weight and blood pressure.    
-
Collect urine sample for urine nitrogen and urine creatinine.  
-
Fasting blood draw (for HgbA1c, plasma glucose, serum insulin, lipid panel, fructosamine, and 
a stored sample).  
-
Provide a test meal consistent with the assigned diet (Control diet arm: 50% carbohydrate, 
15% protein, 35% fat or LoBAG30 diet arm: 30% carbohydrate, 30% protein, 40% fat). In 
addition to the fasting samples, obtain plasma glucose and serum insulin at +30, +60, +90, 
+120, +180 and +240 minutes following the start of the meal. 
-
Collect 3-day food diary.  
 
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-
Collect list of current medications.  
-
Collect end of study compliance survey, Diet Satisfaction Questionnaire, and Diabetes 
Treatment Satisfaction Questionnaire. 
-
Meeting with study dietitian for debriefing and dietary advice for after study completion. 
Dietitian consult may be conducted by phone.  
 
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Protocol Version Date: 03/29/2021 
 

Appendix 2: Updated Procedures During COVID-19 Pandemic 
Due to the risk of coronavirus infec6on and to protect study par6cipants and staﬀ, study procedures will be 
converted to virtual as much as possible during the COVID-19 pandemic. Revised procedures are detailed 
below. These procedures will be u6lized through Sunrise phase 1 and 2, and may be con6nued beyond as 
appropriate according to University of Minnesota guidance.  
Prior to each in-person study visit (the day before and day of) par6cipants will be screened for exposure to or 
symptoms of COVID-19. If exposure or symptoms are reported, the in-person visit will be cancelled.  
Screen. Will usually be done by telephone; may also be done by email or video conference.  
-
In addi6on to previously stated inclusion and exclusion criteria, during the COVID-19 
pandemic par6cipants must have ability to par6cipate in virtual visits, using Zoom or a 
similar HIPAA compliant video conferencing plaYorm. This will require par6cipants to have 
a cell phone with camera or computer with camera and internet connec6on.  
Visit 1. Consent and Screening Visit.  
Part 1: Video conference (40 min). 
-
Review consent document and obtain informed consent using E-consent (delivered and 
signed via REDCap). Complete HIPAA form using same process. Note: If paper consent/
HIPAA is preferred by par6cipant or E-consent is not possible, the consent form will be 
completed in person; no study procedures will take place un6l the consent form is signed.  
-
Collect list of current medica6ons.  
-
If female, determine menopausal status and need for pregnancy test during in-person visit.   
-
Provide instruc6ons for in-person por6on of visit at CRU, including instruc6on to wear a 
mask and to prac6ce social distancing as able. A mask will be provided if needed.  
-
Provide “COVID-19 and Research Par6cipa6on Informa6on Sheet” by mail or email at least 
24 hours in advance of in-person visit.  
-
Provide Greenphire ClinCard FAQ sheet by mail or email. 
Part 2: In person visit at CRU (20 min). 
-
Measure weight and height. 
-
Non-fas6ng blood draw (for HgbA1c, basic metabolic panel, and complete blood count). 
-
Collect urine sample (for baseline urine nitrogen and urine crea6nine, urine microalbumin, and 
pregnancy test if female and premenopausal). 
-
Place Abbod Freestyle Libre CGM. Provide supplies to return the CGM sensor (by mail or in person 
at next visit). 
Provide to par6cipant in-person at visit, or by mail/email/Redcap aEer visit: 
-
Blank 3-day food intake diary  
 
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-
Blank baseline surveys (compliance survey, Diet Sa6sfac6on Ques6onnaire, and Diabetes 
Treatment Sa6sfac6on Ques6onnaire) 
-
Stool collec6on kit and instruc6ons for stool sample collec6on 
-
NCC food amounts booklet for Nutri6on Coordina6ng Center (NCC) diet recall  
-
Ac6graph ac6vity monitor (or provide at visit 2) 
RandomizaBon. No visit needed.  
NCC Diet Recall 
-
Telephone call conducted by the NCC to complete an unannounced 24-hour diet recall (30 
minutes). 
Visit 2. Diet Instruc6on and Baseline Labs (Beginning of Week 1). 
-
Subjects bring to visit, or return by mail or Redcap prior to visit: completed 3-day food diary, 
completed baseline surveys, stool sample, CGM sensor 
-
Provide Ac6graph ac6vity monitor if not already done 
Part 1: In person visit at CRU (4-5 hours; face to face 6me with CRU staﬀ 30 min total) 
-
Measure weight, height and blood pressure 
-
Place IV for use during blood draws 
-
Fas6ng blood draw (for plasma glucose, serum insulin, lipid panel, fructosamine, and a stored 
sample) 
-
Provide a test meal consistent with the assigned diet (control or LoBAG) 
-
In addi6on to the fas6ng samples, obtain plasma glucose and serum insulin at +30, +60, +90, +120, 
+180 and +240 minutes following the start of the meal 
-
Par6cipants will follow CRU protocols for social distancing during this visit. Interac6ons with staﬀ 
will be kept to a minimum. Par6cipants will be asked to wear a mask during the visit, except when 
ea6ng.  
-
Provide handouts for virtual die66an visit (sample menus and recipes consistent with the assigned 
diet). Can alterna6vely be sent by mail/email.  
Part 2: Virtual mee6ng with study die66an for diet instruc6on (1 hour) 
-  
Die66an consult may be conducted by phone or by video conference. Mee6ng can be completed 
between blood draws while par6cipant is at the CRU or from home later, depending on par6cipant 
preference.  
Visit 3. Diet Reinforcement (Week 2).  
 
15
Protocol Version Date: 03/29/2021 
 

Part 1: In person visit at CRU (10 min). Op6on to skip in-person visit if par6cipant and study 
staﬀ agree this is not needed.   
-
Measure weight.  
-
Collect urine sample for urine nitrogen and urine crea6nine.  
-
Return Ac6graph ac6vity monitor if not already done (or return by mail). 
Part 2: Virtual mee6ng with study die66an (30 min). Par6cipant will be at home for this mee6ng.  
-
Reinforcement of diet advice. Adjust caloric intake if needed for goal of weight stability.  
Visit 3.1. (Week 4)  
-
Telephone call or video conference conducted by study inves6gator or coordinator to encourage 
study compliance. 
  
Visit 4. Midpoint Visit (Week 6)  
Surrounding 6me of visit, provide to par6cipant by mail/email/Redcap:  
-
Stool collec6on kit and instruc6ons for stool sample collec6on 
-
Blank 3-day food diary 
-
Blank collec6on form for list of current medica6ons 
-
Blank midpoint compliance survey and Diabetes Treatment Sa6sfac6on Ques6onnaire 
-
Items can be returned in person at visit, by mail, or via Redcap 
Part 1: In person visit at CRU (20 min).  
-
Measure weight and blood pressure. 
-
Fas6ng or non-fas6ng blood draw for HgbA1c, fructosamine, and a stored sample.  
-
Collect urine sample for urine nitrogen and urine crea6nine.  
Part 2: Virtual mee6ng with study die66an (30 min). Par6cipant will be at home for this 
mee6ng.  
-
Reinforcement of diet advice.  
Visit 4.1 (Week 8)   
-
Telephone call or video conference conducted by study inves6gator or coordinator to encourage 
study compliance. 
 
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Protocol Version Date: 03/29/2021 
 

Visit 5. Diet Reinforcement (week 9-10) 
Part 1: In person visit at CRU (20 min).   
-
Measure weight.  
-
Collect urine sample for urine nitrogen and urine crea6nine.  
-
Place Abbod Freestyle Libre CGM. Provide supplies to return the CGM sensor by mail or in 
person at next visit 
-
Provide Ac6graph ac6vity monitor (or send by mail) 
Part 2: Virtual mee6ng with study die66an (30 min). Par6cipant will be at home for this mee6ng.  
-
Reinforcement of diet advice. 
Visit 5.1. (Week 11)  
 
-
Telephone call or video conference conducted by study inves6gator or coordinator to encourage 
study compliance. 
NCC Diet Recall  
-
Telephone call in week 9-12 conducted by the NCC for an unannounced 24-hour diet recall (30 
minutes). 
Visit 6. Final Visit (end of Week 12)  
Provide to par6cipant for comple6on prior to visit 6: 
-
Blank 3-day food intake diary  
-
Blank ﬁnal surveys (compliance survey, Diet Sa6sfac6on Ques6onnaire, and Diabetes Treatment 
Sa6sfac6on Ques6onnaire) 
-
Stool collec6on kit and instruc6ons for stool sample collec6on 
-
Ac6graph ac6vity monitor  
-
Blank collec6on form for list of current medica6ons 
-
Items can be returned in person at visit, by mail, or via Redcap 
Part 1: In person visit at CRU (4-5 hours; face to face 6me with staﬀ 30 min total) 
-
Measure weight and blood pressure 
-
Collect urine sample for urine nitrogen and urine crea6nine.  
-
Place IV for use during blood draws 
-
Fas6ng blood draw (for HgbA1c, plasma glucose, serum insulin, lipid panel, fructosamine, and a 
stored sample).  
-
Provide a test meal consistent with the assigned diet (control or LoBAG) 
 
17
Protocol Version Date: 03/29/2021 
 

-
In addi6on to the fas6ng samples, obtain plasma glucose and serum insulin at +30, +60, +90, +120, 
+180 and +240 minutes following the start of the meal 
-
Par6cipants will follow CRU protocols for social distancing during this visit. Interac6ons with staﬀ 
will be kept to a minimum. Par6cipants will be asked to wear a mask during the visit, except when 
ea6ng.  
Part 2: Op6onal virtual mee6ng with study die66an for debrieﬁng and dietary advice for aEer study 
comple6on (30 min).  
-
Die66an consult may be conducted by phone or by video conference. Mee6ng can be 
completed between blood draws while par6cipant is at the CRU or from home.  
Part 3: Video or phone mee6ng with study inves6gator or coordinator for closeout, and to ensure all 
study procedures complete (10 min).  
  
Study CompleBon  
 
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Protocol Version Date: 03/29/2021