The Effect of Glucagon on Rates of Hepatic Mitochondrial Oxidation and Pyruvate Carboxylase Flux in Man Assessed by Positional Isotopomer NMR Tracer Analysis (PINTA)
Phase: PHASE1
Conditions: Healthy Participants
Sponsor: Yale University

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Effective Date: 25 April 2013     
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Investigator Studies Program 
Review Committee (MISP-RC)
IIS Clinical Concept Form
All fields are required, an incomplete form will be returned to the submitter.  If a field is not completed, please note the 
reason.
Proposed Study Title
Study Title:
The effect of glucagon on rates of hepatic mitochondrial oxidation and pyruvate 
carboxylase flux in man assessed by Positional Isotopomer NMR Tracer Analysis 
(PINTA)
Request Date:
July 1, 2019
Principal Investigator Contact Information
Names: 
Kitt Falk Petersen, M.D. 
Title:
 Professor of Medicine
Address 1
 Yale University School of Medicine
Address 2
 333 Cedar St., P.O. Box 208020
City, ST, Zip
 New Haven, CT  06520-8020
Phone/Fax:
 (203) 785-5447
E-mail:
 kitt.petersen@yale.edu, 
Institution Contact Information
Name: 
 Andrea Lozano
Address 1
Office of Sponsored Projects
Address 2
150 Munson Street, PO Box 208327
City, ST, Zip
 New Haven, CT 06520-8327
Phone/Fax:
 203-785-6878
website
 https://your.yale.edu/research-support/office-sponsored-projects
Contracting Information (if applicable)
Name: 
 N/A
Study Information
Indication
 N/A
Phase:
 N/A
Number of Subjects:
12
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Background and Rationale 

Provide background on unanswered question(s) the study is attempting to answer (do not exceed one page)
While it is well-established that alterations in the portal vein insulin/glucagon ratio play a major role in 
causing dysregulated hepatic glucose metabolism in type 2 diabetes (T2D), the cellular and 
molecular mechanisms by which glucagon promotes alterations in hepatic glucose production and 
mitochondrial oxidation remain poorly understood. This is borne out by the fact that both glucagon 
agonists and glucagon antagonists are being developed to treat T2D, with unclear mechanisms of 
action. 
Glucagon has long been considered to be one of the major drivers of hyperglycemia in T2D, and 
glucagon-blocking therapies have been pursued as adjuncts to therapy for both type 1 and type 2 
diabetes. Glucagon antagonism using either antibodies against glucagon or its receptor, small 
molecule antagonists of the glucagon receptor, and antisense oligonucleotides to knock down 
expression of the glucagon receptor have all shown promising glucose-lowering effects in humans 
and in animal models of T2D. However, concerns have been raised regarding the potential of these 
agents to increase liver enzymes by an unknown mechanism. Conversely, a dual glucagon-like 
peptide-1/glucagon receptor agonist has recently been shown in rodents and non-human primates to 
lower blood glucose concentrations, associated with increased energy expenditure and weight loss. 
Taken together, these data suggest a potential role for glucagon to promote hepatic mitochondrial fat 
oxidation and suggest that a lack of glucagon action may suppress hepatic fat oxidation and 
predispose to ectopic hepatic lipid accumulation and non alcoholic fatty liver disease (NAFLD). 
 
The impact of glucagon on hepatic mitochondrial oxidative metabolism in humans is unknown but 
previous studies have suggested a capacity for glucagon to increase liver fatty acid metabolism in 
vitro, and to stimulate hepatic fat oxidation in awake dogs. As predicted by these data, multiple 
investigators have observed increases in CO2 generation, O2 consumption, and activity of the 
respiratory transport chain in response to glucagon stimulation, consistent with increased 
mitochondrial tricarboxylic acid (TCA) cycle flux; however, the mechanism by which glucagon 
stimulate hepatic mitochondrial oxidation and its impact on hepatic mitochondrial oxidation in vivo are 
unknown. In addition we will assess the effects of a physiological increase in plasma glucagon 
concentrations on rates of hepatic ketogenesis assessed by an infusion of 13C4 OHB (Perry et al. 
Cell Metabolism 2017). We have recently shown that allosteric regulation of pyruvate carboxylase 
by changes in hepatic acetyl-CoA is a key mechanism by which insulin (Perry et al. Cell 2015) and 
glucagon (unpublished results) regulate hepatic gluconeogenesis and that rates of OHB turnover 
are an excellent non invasive surrogate for hepatic acetyl-CoA content (Perry et al. Cell Metabolism 
2017).
This proposal will directly assess the effect of glucagon on rates of hepatic mitochondrial oxidation 
and pyruvate carboxylase flux in humans for the first time using Positional Isotopomer NMR Tracer 
Analysis (PINTA) (Perry et al. Nature Communications 2017). The results of these studies have 
important implications for the possibility of intervening in the pathogenesis of NAFLD-associated 
hepatic insulin resistance and T2D via chronic dual glucagon-like peptide-1/glucagon receptor 
agonism and provide an important rationale for why a dual agonist might be more efficacious for 
treating NAFLD/NASH/T2D than GLP-1 alone.
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Objectives

List the objectives to correspond directly with the listed hypotheses:
To examine the effects of glucagon on hepatic glucose and fat metabolism in vivo, we plan to apply a 
novel Positional Isotopomer NMR Tracer Analysis (PINTA) method to quantify rates of hepatic 
mitochondrial oxidation (VCS) and pyruvate carboxylase flux (VPC), which has been cross-validated in 
awake rodents and humans (Perry et al. Nature Communications 2017). In preliminary rodent 
studies we have found that glucagon stimulates intrahepatic lipolysis through an InsP3R-I-dependent 
process, leading to increases in hepatic acetyl-CoA content, which allosterically activates pyruvate 
carboxylase activity and flux (VPC), and that this phenomenon explains its acute, transcription-
independent effect to acutely stimulate hepatic gluconeogenesis in vivo (unpublished results). In 
addition, using PINTA analysis we found that glucagon stimulates hepatic mitochondrial oxidation 
(Vcs) through InsP3R-I-mediated calcium signaling in awake mice, and that this process can be 
exploited by short-term continuous glucagon treatment leading to two-fold increases in hepatic 
mitochondrial fat oxidation (VFAO), which in turn results in large reductions in hepatic steatosis and 
marked improvements in glucose tolerance through reversal of hepatic insulin resistance in a high fat 
fed rat model of NAFLD.
Hypothesis 

List the clinical Hypotheses in order of priority:
1. A physiological increase in plasma glucagon concentrations will promote a significant increase 
in rates of hepatic mitochondrial oxidation (VCS) in healthy humans.
2. A physiological increase in plasma glucagon concentrations will promote a significant increase 
in rates of hepatic pyruvate carboxylase flux (VPC) in healthy humans.
3. A physiological increase in plasma glucagon concentrations will promote a significant increase 
in rates of 13C4 -OHB turnover (hepatic ketogenesis) in healthy humans.
Study Design/Clinical Plan

Provide a concise overview stating the type of experimental design
The effects of a physiological increase in plasma glucagon on rates of hepatic mitochondrial 
oxidation and pyruvate carboxylase flux will be examined in 12 healthy subjects (ages 21-65) using 
Positional Isotopomer NMR Tracer Analysis (PINTA) (Perry et al. Nature Communication 2017). 
Briefly rates of hepatic mitochondrial oxidation (VCS) and hepatic pyruvate carboxylase flux (VPC) will 
be assessed in 12 healthy overnight fasted subjects by PINTA after a three-hour infusion of glucagon 
or saline. The glucagon infusion [3 ng/(kg-min)] will be designed to increase peripheral and portal 
vein plasma glucagon concentrations 3-4 fold. The effects of a physiological increase in plasma 
glucagon on rates of hepatic ketogenesis will also be assessed using an infusion of 13C4 OHB 
(Perry et al. Cell Metabolism 2017). 
Rates of hepatic pyruvate carboxylase flux  (VPC)/citrate synthase flux  (VCS)  by  PINTA: 
Subjects (n=12) will be studied by PINTA under 2 conditions: 1) at 0700 following an overnight 
(12h) fast and a 3h saline infusion (Control), 2) at 0700 following an overnight (12h) fast and a 3h 
glucagon infusion @ [3 ng/(kg-min)]  (Glucagon Rx). Briefly, on the morning of the study an IV line 
will be inserted into an antecubital vein for infusions and second IV line will be inserted 
retrogradely into a hand vein for blood collections. The hand will be warmed in a ‘hot box’ 
to arterialize the blood. After collection of baseline blood samples a 3h infusion of tracers as 
described below will be started. Relative rates  of VPC/VCS  will be assessed  using  a constant  
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Effective Date: 25 April 2013     
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infusion  of [3-13C]  lactate  (8.7  µmol/[Kg-min],  99%  APE)  and  rates  of glucose  production  
will be measured  using  a  primed/constant  infusion  of  [2H7]glucose  (42  mg/m2   -  0.84  
mg/[m2min],  99%  APE) (Perry et al. Nature Communication 2017). Rates of hepatic 
ketogenesis will be measured using a constant infusion of [ 1 3C 4]βOHB [10µg/(Kg-min), 99% 
APE] as previously described (Perry et al. Cell Metabolism 2017). This protocol has been 
approved the Yale Human Investigation Committee (#20997).
GC/MS  Method   for  Quantifying [13C4]β OHB  turnover:  Analysis  of  the 13C-enrichment  in  
β OHB  will  be performed by GC-MS. Samples (100 μL plasma) will be dried under N2  gas and 
derivatized with 200 μL n- butanol 4N HCl, then heated for 60 min at 65°C and dried under N2 
gas. The samples will next be reacted with100  μL  of  trifluoroacetic  acid  (Thermo  Fisher  
Scientific)-methylene   chloride  (Sigma)  (1:7).  [13C4]βOHB enrichment will be measured by GC-
MS (CI mode, m/z 257 [m+0] and 261 [m+4]). 
Whole body  energy  expenditure, VO2,  VCO2,  respiratory  quotient  assessed by  indirect 
calorimetry: Rates of total-body  glucose  and  lipid  oxidation  and  energy  expenditure  will  
be  calculated  from  rates  of  O2 consumption and CO2 production measured during continuous 
indirect calorimetry.
This trial is registered at CT.gov: NCT 03965130
References
1.
Perry RJ, Peng L, Cline GW, Petersen KF, Shulman GI. A non-invasive method to assess hepatic 
acetyl-CoA in vivo. Cell Metab. 2017; 25(3) 749-756. PMCID: PMC5342911. PMID: 28111213.
2.
Perry RJ, Peng L, Cline GW, Butrico GM, Wang Y, Zhang XM, Rothman DL, Petersen KF, Shulman GI. 
Non-invasive assessment of hepatic mitochondrial metabolism by positional isotopomer NMR tracer 
analysis (PINTA). Nature Communications, 2017; 9 (1) 1-9. PMCID: PMC5630596, PMID:  28986525.
3.
Perry RJ, Wang Y, Cline GW, Rabin-Court A, Song JD, Dufour S, Zhang XM, Petersen KF, Shulman GI. 
Leptin mediates a glucose-fatty acid cycle to maintain glucose homeostasis in starvation. Cell. 2018; 
172 (1-2) 234-248. PMCID: PMC5766366, PMID: 29307489.
Treatment

List the clinical dosage/dosage form, route, and dose regimen:
Glucagon will be infused intravenously at rate of 3 ng/(kg-min) for three hours.
Collateral Research 

Include biomarkers, PK, etc. 
Plasma samples will be collected at the end of each infusion study and banked for future 
metabolomic analyses to determine if any plasma metabolites track with glucagon-stimulated rates of 
hepatic mitochondrial oxidation and/or pyruvate carboxylase flux. 
Statistical Plans

Include justification for clinical sample size and primary hypothesis testing:
A group size of 12 subjects was determined based on power calculations to detect a moderate to 
large difference in the effects of glucagon on rates of hepatic mitochondrial oxidation (VCS). The key 
endpoints will be to determine if glucagon treatment results in significant differences in: 1) rates of 
hepatic mitochondrial oxidation, 2) rates of hepatic pyruvate carboxylase flux (VPC), 3) rates of 
hepatic glucose production, and 4) rates of hepatic OHB turnover. Statistical differences in these 
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Effective Date: 25 April 2013     
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parameters (within the same individuals) for saline vs. glucagon treatment will assessed by paired t-
tests.
Budget Summary

Please be sure to complete budget template (excel document)
Total Amount Requested:
(Include overhead)
$1,295,423 (including 30% Yale overhead)
Additional sources of 
funding required? (Yes/No)
If Yes, please be specific.
No
Timelines and Study Plans
Number of Sites:
 1
Site Names:
Yale Center for Clinical Investigation, Hospital Research Unit
Study Start Date:
 July 1, 2019
Study End Date:
June 30, 2020
Number of Subjects:
10
First Patient In Date:
July 1, 2019
Last Patient Out Date:
May 30, 2020
Enrollment Period in 
Months:
11
Publication Plan
Where are you planning to 
submit for publication? 
(journals, etc):
 Cell Metabolism, Journal of Clinical Investigation, Nature Medicine, PNAS
Are you planning to present 
your data at a scientific 
meeting?
 
Please list your target date 
for submission of 
publication.
Approximately 2020
Drug Supply Information
Drug Supplies Required 
(Yes/No)?  
 No
List Drug Supplies and 
Amount Required:
Drug Name:
Amount:
List Drug Supplies and 
Amount Required:
Drug Name:
Amount:
Placebo Required 
(Yes/No)?
No
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Additional Sources of Drug 
Supply (Yes/No).  If Yes, 
please specify
No
APPROVED BY THE YALE UNIVERSITY IRB 6/5/2019