Dietary Ketosis a Metabolic Sister to Calorie Restriction (CR): Fatty Acids Activate AMPK Energy Circuits Modulating Global Methylation Via the SAM/SAH Axis
Phase: NA
Conditions: Mild Cognitive Impairment, Metabolic Syndrome
Sponsor: Bristlecone Health, Inc.

 
 
 
 
 
 
 
 
 
 
STUDY PROTOCOL 
Title: Dietary Ketosis a Metabolic Sister to Calorie Restriction (CR): Fatty acids activate 
AMPK energy circuits modulating global methylation via the SAM/SAH axis 
NCT03319173 
15 September 2017 
 
 
 
 
 
 
 
 
 
 
 

STUDY PROTOCOL 
Title of Study: Dietary Ketosis a Metabolic Sister to Calorie Restriction (CR): Fatty acids 
activate AMPK energy circuits modulating global methylation via the SAM/SAH axis 
Principle Investigator: Dr. Kelly J. Gibas, CFMP, LPCC 
           
• Protocol Number: Bristlecone-001 
• Protocol Date: 09/15/2017 – 05/15/2018 
• Version #1 
• Investigators: Dr. Kelly J. Gibas (Principle Investigator) 
                                               Dr. Julie A. Gomer (Sub-investigator)  
• Bristlecone Health, Inc. 
 
 
13700 Reimer Drive N., Suite 220 
  
 
Maple Grove, MN  55311 
 
 
763-424-2474 
 
 
www.bristleconemedical.com 
• Source of external funding: N/A 
• Hypothesis or Study Synopsis: 
Nutritional epigenetics denotes gene–diet interactions and highlights the modulatory role of 
nutrition in aging and age-related diseases such as cancer, CVD, diabetes and neurodegenerative 
disorders. Nutrients are a source of epigenetic modification; they are able to regulate the 
placement of histone modifiers distinguishing phenotype from genotype. The energy status of the 
cells (fed or fasted) modulates the regulation of global DNA methylation via the S-
adenosylmethionine (SAM), the methyltransferase inhibitor, S-adenosylhomocysteine (SAH) 
axis and whole blood histamine levels. Insulin resistance and hyperinsulinemia dysregulate 
cellular signals leading to metabolic inflexibility. Chronic elevations in insulin with long-
standing impairments in glucose delivery are associated with profound changes in epigenetic 
patterns due to over-activation of the mTOR kinase pathway and repression of AMPK. Dietary 
ketosis is known to change the metabolic status of the cells by increasing the AMP/ATP ratio. 
AMPK activation adapts rRNA synthesis away from growth/biosynthesis and toward ATP 
availability and utilization, thereby, attenuating the progression of hypo-metabolic diseases in the 
body and the brain at the level of the genome.   
 
The study will explore whether early stage memory loss (SMC & MCI) and the comorbidity of 
Metabolic Syndrome are symptomatic of peripheral and cerebral cellular hypo-metabolism 
induced by chronic insulin resistance. We will attempt to show that consequential to systemic 
hyperinsulinimia, aberrant crosstalk between the mitochondria and nuclear genome results in the 
dysregulation of the regulatory kinases mediating metabolic state and intracellular/extracellular 
signaling: mTOR and AMPK. The suppression of AMPK signals with chronic overexpression of 

mTOR signaling will adapt rRNA synthesis away from nutrient availability and toward ATP 
consuming processes: the biosynthesis of cholesterol, triglycerides, glycogen with inhibition of 
fatty acid oxidation, histone acetylation with a down-regulation of NAD+ and SAHH cofactors 
leading to global DNA hypo-methylation and local hyper-methylation, suppression of the 
SAM/SAH ratio, the inhibition of SIRT (sirtuin) expression and normalized whole blood 
histamine levels. These epigenetic shifts mediate global metabolic inflexibility by channeling 
fuel substrates toward cytosolic, substrate level phosphorylation (SLP) via over expression of the 
glycolytic enzymes including PDK (pyruvate dehydrogenase kinase) and away from 
mitochondrial oxidation mediated by the suppression of PDC (pyruvate dehydrogenase 
complex), the major regulatory gateway of metabolism between glycolysis and citric acid cycle. 
We will attempt to show that activation of the AMPK pathway via induced and controlled 
dietary ketosis will inhibit mTOR signaling away from the biosynthesis of energy and SLP 
toward the generation of ATP by increasing the cellular AMP/ATP ratio, thus regulating 
oxidative metabolic signals and attenuating global, cellular hypo-metabolism evidenced by 
marked reductions in lipid synthesis and LP-IR score (particle concentration and size), HgA1c, 
fasting insulin/HOMA-IR, blood ketones, fasting triglycerides together with epigenetic 
regulation of DNA methylation status  including normalized whole blood histamine levels and 
homo-cysteine regulation. Improvement in cerebral glucose metabolism and corresponding 
diagnosis of SMC/MCI will be assessed by the objective changes in the outcome measures of 
MoCA, BVMT-R (Brief Visual Memory Test-Revised) and Rey Auditory Verbal Learning Task 
(RAVLT), administered at baseline and weeks 2/4/6/8/10/12.   
 
Research Question:  Are selective memory complaints (SMC), mild cognitive impairment 
(MCI) and comorbidity of Metabolic Syndrome symptomatic of peripheral and cerebral hypo-
metabolism with corresponding epigenetic shifts in global DNA hypo-methylation/histone 
acetylation away from nutrient availability and toward biosynthesis with up-regulation of 
substrate level phosphorylation (SLP) initiated by sustained metabolic inflexibility 
(hyperinsulinemia), over-activation of the mTOR kinase pathway, and repression of 
mitochondrial oxidation via inhibition of pyruvate dehydrogenase complex (PDC)?  
• Background: 
 
 
Dr. Kelly J. Gibas, CFMP, LPCC 
 
 
Doctor of Clinical Behavior Sciences  
 
 
Assistant Professor: Human Bioenergetics & Applied Health Science at Bethel  
 
 
 
       University 
 
 
Bristlecone Health, Inc.: Founder/CEO/Clinical Director 
 
 
Study interests: Metabolome; the study of impaired glucose  
 
 
 
 
metabolism an cerebral/somatic epigenetic histone modulation leading  
 
 
 
to chronic degenerative diseases 
 
 
 
 
Recent publications:  
M.K Gibas, K.J. Gibas, Induced and controlled dietary ketosis as a regulator of obesity  

and metabolic syndrome pathologies, Diab Met Syndr: Clin Res Rev (2017), 
http://dx.doi.org/10.1016/j.dsx.2017.03.022  (RCT, March 2017) PMID:28433617 
 
Gibas, K.J (2017). The Starving Brain: Overfed meets undernourished in the pathology of mild cognitive 
impairment (MCI) and Alzheimer’s disease (AD). Neurochemistry International, 
https://doi.org/10.1016/j.neuint.2017.09.004. PMID: 28899812 
Gibas, K.J. The Retrograde Signal: Glucose dependency marks the cancerous phenotype. Austin Diabetes 
Research, Austin Diabetes Res. 2017; 1(1): 1065. 
Dahlgren K, Gibas KJ. Ketogenic diet, high intensity interval training (HIIT) and memory training in the 
treatment of mild cognitive impairment: A case study. Diabetes & Metabolic Syndrome: Clinical 
Research & Reviews. 2018. doi:10.1016/j.dsx.2018.04.031. 
Brown D, Gibas KJ. Metabolic syndrome marks early risk for cognitive decline with APOE4 gene 
variation: A case study. Diabetes & Metabolic Syndrome: Clinical Research & Reviews. 2018. 
doi:10.1016/j.dsx.2018.04.030. 
Gibas KJ, Halikas A. AMPK induced memory improvements in the diabetic population: A Case 
Study. Diabetes & Metabolic Syndrome: Clinical Research & Reviews. 2018. 
doi:10.1016/j.dsx.2018.04.033. 
Gibas, K. (December 2018). Mouth breather? Enduring patterns of hypocapnia induce cerebral hypoxia 
and consequential cognitive impairments mediated by aberrant HIF-1 survival signals common 
to the cell danger response. Poster presentation at Cell Symposia: Metabolites as Signaling 
Molecules Conference, 2018, Seattle, WA. http://www.cell-symposia.com/metabolites-
2018/conference-program.asp 
Morrill SJ, Gibas KJ. Ketogenic diet rescues cognition in ApoE4 patient with mild Alzheimer’s disease: 
A case study. Diabetes & Metabolic Syndrome: Clinical Research & Reviews. 2019;13(2):1187-
1191. doi:10.1016/j.dsx.2019.01.035. 
Cox N, Gibas S, Salisbury M, Gomer J, Gibas K. Ketogenic diets may reverse Type II diabetes and 
ameliorate clinical depression: A case study. Diabetes & Metabolic Syndrome: Clinical Research 
& Reviews. 2019. doi:10.1016/j.dsx.2019.01.055. 
Stoykovich S, Gibas K. APOE ε4, the door to insulin-resistant dyslipidemia and brain fog? A case 
study. Alzheimers & Dementia: Diagnosis, Assessment & Disease Monitoring. 2019;11:264-269. 
doi:10.1016/j.dadm.2019.01.009. 
Gibas, K. & Stoykovich, S. (April, 2019). ApoE4, the door to insulin-resistant dyslipidemia and brain 
fog? A case study. Poster presentation at American Diabetes Association/WCITD – 2019. New 
York, NY. 
https://cmoffice.kenes.com/cmsearchableprogrammeV15/conferencemanager/programme/personi
d/anonymous/WCITD19/normal/b833d15f547f3cf698a5e922754684fa334885ed#!abstractdetails/
0000394330 
 

Study Goals and Objectives 
Primary objectives:  
1. Improve cerebral and peripheral substrate oxidation as measured by cognitive 
assessments (MoCA, BVMT-R & RAVLT) and improved blood lipids (reduction in 
small LDL particle size) 
2. Normalizing the SAM/SAH ratio and whole blood histamine levels to restore global 
DNA methylation for disease prevention 
3. Restoration and balancing of the AMPK/mTOR pathways for disease prevention 
4. Alleviate insulin resistance in order to reverse T2DM 
5. Restore cerebral glucose metabolism in order to prevent neuron death 
6. Prevent cardiovascular disease by restoring metabolic flexibility through fatty acid 
oxidation 
Secondary objectives:   
1. Improved self-efficacy; empowerment to normalize blood lipids through nutritional 
interventions 
2. Reduction in depression and anxiety associated with metabolic syndrome 
3. Reduction in weight, BMI, body fat mass, HgA1c and normalized blood pressure  
Subject Selection: 
• The study subjects are adults (18-80) referred by their physician and previously 
diagnosed with MetS and/or T2DM as measured by possessing at least 2 of the 
following physiological measures: type II diabetes, BMI >30, HgA1c > 5.7, 
waist/height ratio >.6, fasting glucose > 125. All subjects will volunteer for the 
study after being referred by their healthcare provider; they will be randomly 
assigned to one of two groups: experimental group with induced and controlled 
dietary ketosis or control group with standard American diet.  Participants in both 
groups will also be required to play PEAK brain training games on iPhone, iPad 
or Android devices for 120 minutes per week.   
• The study will include at least 30 subjects (15 per group) but no more than 80 
subjects (40 per group). 
• Men and women only (ages 18-80) 
  
Inclusion/Exclusion criteria: 
 
                  Inclusion criteria:  
• Male or Female (age 18-80) 
• Previously diagnosed with MetS and/or T2DM as measured by possessing at least 
2 of the following physiological measures: type II diabetes, BMI >30, HgA1c > 
5.7, waist/height ratio >.6, fasting glucose > 125 
• Subjective Memory Complaints (SCM) – Subjects score > 3 ‘yes’ answers on the 
Subjective Memory Complaints Questionnaire at the initial visit 
• Previously diagnosed with Mild Cognitive Impairment (MCI) 
                  Exclusion criteria: 

• Previously diagnosed with Alzheimer’s disease (AD), dementia or Parkinson’s 
disease  
 
 
 
Study procedures/Research method 
 
• The duration of subjects’ participation will be 12 weeks.  They will be asked to 
meet with Dr. Gibas or Dr. Gomer each week at Bristlecone Health, Inc. for 
approximately 20-30 minutes.  During that time, they will receive cognitive 
assessments (MoCA, BVMT-R, and Rey Auditory Verbal Learning Task), which 
are memory tests.  They will be conducted at Bristlecone Health, Inc. on weeks 0 
and 12.  Blood ketones and fasting triglycerides will be measured at Bristlecone 
Health, Inc. on weeks 0, 3, 6, 9 and 12.  Participants will also receive weekly 
weight and body fat measurements. 
• Pre/post blood labs will be drawn to measure biomarker changes associated with 
MetS on weeks 0 and 12 at a North Memorial Reference Laboratory. The 
following blood labs will be assessed: HgA1c, fasting insulin, fasting glucose, 
NMR particle size testing and methylation profile, SAM/SAH ratio testing and 
whole blood histamine.  There are 21 North Memorial Reference Laboratories 
throughout the metro area for convenience.    
• Subjects will be randomly assigned to either an experimental or control group in 
the order of referral (e.g., subject number one assigned to experimental group; 
subject number two assigned to control group; subject number three assigned to 
control group, and so on).  Regardless of group assignment, the subject will 
receive the aforementioned cognitive and blood lab assessments. 
• Subjects in the experimental group will receive clinically regulated meal plans 
designed to facilitate prolonged benign dietary ketosis (BDK) in order to regulate 
glucose with restored insulin sensitivity focused at reversing the impaired 
capacity to switch between fat and carbohydrate oxidation.  Subjects in the 
control group will follow their current dietary protocol (Standard American Diet-
SAD).  Both groups will play PEAK brain training game on iPhone, iPad or 
Android devices for 120 minutes per week. 
     
 
RESEARCH METHOD: 
 
 
Analyses of variance (ANOVA) will be run to assess differences   
 
 
 
between the experimental and control group. The ANOVA will be run  
 
 
 
on both groups measuring baseline and post program (week 0 and   
 
 
 
week 12) HgA1c, fasting insulin, fasting glucose, NMR particle size  
 
 
 
testing and a methylation profile, which includes: SAM/SAH ratio test and whole  
 
 
blood histamine.  
 
 
An ANOVA will be run on both groups measuring   
 
 
 
 
 
blood ketones and fasting triglycerides at weeks 0, 3, 6, 9 and 12. The  
 

 
 
ANOVA will also be run on both groups measuring cognitive  
 
 
 
 
measures using the Rey Auditory Verbal Learning Task, BVMT-R  
 
 
 
and MoCA assessments at weeks 0 and12. Mean scores will be  
 
 
 
 
used to assess significant differences between the two groups. Further  
 
 
 
analyses of possible treatment effects will be examine within-group  
 
 
 
differences using a paired sampled t-test.   
Risk / Safety Information 
• There are few foreseeable risks associated with this study.  No costs are 
associated with participation nor will the subjects’ decision to participate affect 
their employment/medical care or insurance status.  Discomfort associated with 
study participation may include short-term general malaise commonly 
associated with dietary changes (if the subject is in the experimental group). 
Discomfort during blood draws at North Memorial Reference Laboratories may 
also occur.  If subjects experience any discomfort, they will be instructed to 
inform the phlebotomist immediately.  This study is considered to have minimal 
risk. 
• The benefits which may reasonably be expected to result from this study are 
improved memory as measured by cognitive assessments (MoCA, BVMT-R, 
and Rey Auditory Verbal Learning Task), reversal of MetS, improved blood 
lipid biomarkers, decreased HgA1c, increased blood ketone levels, and reduced 
weight and body fat mass. 
 
Monitoring and reporting of Adverse Events/Serious Adverse Events 
• Subjects will receive instructions to contact the principal investigator or sub-
investigator for nutritional counsel if they experience general malaise or any 
discomfort when changing dietary protocol. 
• Subjects will receive instructions to inform the phlebotomist at North Memorial 
Reference Laboratory if they feel any discomfort during blood collection.  
• If a subject presents with an adverse effect from following the study protocol, 
they will be referred back to their primary care physician in order to determine if 
continued study participation is recommended.  Subjects will only be allowed to 
continue with the study with written permission from their primary care provider. 
• Any unanticipated problems will be reported to the IRB within (10) calendar days 
of being reported to the investigator. 
Study Oversight 
• The principle investigator (Dr. Kelly J. Gibas) will provide all oversight for the 
project and supervision all study activities.   
• The only other person who will have access to subjects is sub-investigator (Dr. 
Julie A. Gomer). 
• The Investigator will ensure that all study staff are adequately trained in the study 
protocol 

• Both Dr. Gibas and Dr. Gomer have received NIH certification:  “Protecting 
Human Research Participants” 
Certificate of Completion 
The National Institutes of Health (NIH) Office of Extramural Research certifies that 
Kelly Gibas successfully completed the NIH Web-based training course 
"Protecting Human Research Participants". 
Date of completion: 07/25/2017. 
Certification Number: 2440977. 
 
Certificate of Completion 
The National Institutes of Health (NIH) Office of Extramural Research certifies that 
Julie Gomer successfully completed the NIH Web-based training course 
"Protecting Human Research Participants". 
Date of completion: 07/30/2017. 
Certification Number: 2442927. 
• Data Management 
o Who will be collecting, analyzing the data?  Dr. Gibas & Dr. Gomer 
o Do you plan to have an independent data analysis?  Yes, through the 
University of Minnesota (Aaron Rendahl, PhD and Yiwen Sun, PhD) 
o Data retention (i.e. how long so you plan to maintain records for this 
study) – 2 years post study 
• IRB Review / Ethics / Informed Consent 
o Explain your informed consent process.  
 
1. Subjects will be given the informed consent during an individual 
intake meeting (after being referred by their primary care provider). 
2. Subjects will be provided with the opportunity to take the informed 
consent home to discuss with their families. 
3. Dr. Gibas or Dr. Gomer will explain the informed consent and answer 
any questions. 
4. Once the subject signs the consent, they will receive a copy of the 
signed consent for their records. 
• Confidentiality 
o Discuss the procedures that will be used to maintain the confidentiality of 
the research data. Specifically, how will data be stored to ensure that 
it is secure and remains confidential?   

• Research data will be used for the purposes of this study only.  Copies of 
medical and clinical measures will be stored in a locked file at Bristlecone 
Health, Inc. and will only be accessed by Dr. Kelly J. Gibas and Dr. Julie A. 
Gomer (co-investigator).  If a subject decides to withdraw from the study, 
his/her confidential medical data and clinical measures will be destroyed within 
24 hours of receipt of the written request to withdraw from the study 
 
• Data collection and reporting for the purposes of this study will remain 
confidential and subject identity will not be identifiable.  Each subject will be 
given a unique ID code for the purposes of data analysis.  Upon completion of 
the study, subjects have 8 weeks to obtain a copy of their clinical data measures.  
The data will be retained for 2 years post study. 
• Data may be published in certain journals. Upon publication of the 
aforementioned study, all protected health information (PHI) will be protected 
and subjects’ identities will not be disclosed in the publication or any other forum.