The Effect of Glucagon on Rates of Hepatic Mitochondrial Oxidation in Man Assessed by PINTA

Study Overview
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Brief Summary
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It is well established that alterations in the portal vein insulin:glucagon ratio play a major role in the dysregulated hepatic glucose metabolism in type 2 diabetes but the molecular mechanism by which glucagon promotes alterations in hepatic glucose production and mitochondrial oxidation remain poorly understood. This is borne out of the fact that both glucagon agonists and antagonists are being developed to treat type 2 diabetes with unclear mechanisms of action. This study will directly assess rates of mitochondrial oxidation and pyruvate carboxylase flux for the first time in humans using PINTA analysis as well as the effects of glucagon. The results will have important implications for the possibility of intervening in the pathogenesis of non alcoholic fatty liver and type 2 diabetes via chronic dual GLP-1/glucagon receptor antagonism and provide an important rationale for why a dual agonist may be more efficacious for treatment of non alcoholic fatty liver and T2D than GLP-1 alone.

Detailed Description
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Objectives: To examine rates of hepatic mitochondrial oxidation in healthy volunteers with liver lipid less than 2%, participants with NAFLD/NASH and participants with Type 2 Diabetes (T2D) to assess whether hepatic mitochondrial fat oxidation is altered in people with NAFLD/NASH and T2D. To examine the effects of glucagon on hepatic glucose and fat metabolism in vivo, this study will apply a novel Positional Isotopomer NMR Tracer Analysis (PINTA) method to quantify rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux, which has been cross-validated in awake rodents and humans (Perry et al. Nature Communications 2017). Preliminary rodent studies 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, and that this phenomenon explains its acute, transcription-independent effect to acutely stimulate hepatic gluconeogenesis in vivo (unpublished results). In addition, using PINTA analysis it has been shown that glucagon stimulates hepatic mitochondrial oxidation through 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, 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 non alcoholic fatty liver. Hypothesis: Rates of hepatic mitochondrial function is similar in people with no liver fat and people with NAFLD/NASH and T2D. A physiological increase in plasma glucagon concentrations will promote a significant increase in rates of hepatic mitochondrial oxidation in healthy humans and NAFLD/NASH and T2D. A physiological increase in plasma glucagon concentrations will promote a significant increase in rates of hepatic pyruvate carboxylase flux in healthy humans and NAFLD/NASH and T2D. A physiological increase in plasma glucagon concentrations will promote a significant increase in rates of 13C4 β-hydroxybutyrate turnover (hepatic ketogenesis) in healthy humans and NAFLD/NASH and T2D. Study Design - Clinical Plan: The effects of a physiological increase in plasma glucagon on rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux will be examined in groups of 25 healthy participants (ages 21-65) control, NAFLD/NASH and T2D using Positional Isotopomer NMR Tracer Analysis (PINTA) (Perry et al. Nature Communication 2017). Briefly rates of hepatic mitochondrial oxidation and hepatic pyruvate carboxylase flux will be assessed in 12 healthy overnight fasted participants by PINTA after a three-hour infusion of glucagon or saline. The glucagon infusion 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 β-betahydroxybutyrate (Perry et al. Cell Metabolism 2017). Rates of hepatic pyruvate carboxylase flux /citrate synthase flux by PINTA: Participants (n=12) will be studied by PINTA under 2 conditions: 1) following an overnight fast and a 3 hour saline infusion (Control), 2) following an overnight fast and a 3 hour glucagon infusion. Briefly, after collection of baseline blood samples a 3 hour infusion of tracers as described below will be started. Relative rates of pyruvate carboxylase to citrate synthesis flux will be assessed using a constant infusion of [3-13C] lactate and rates of glucose production will be measured using an infusion of [2H7]glucose (Perry et al. Nature Communication 2017). Rates of hepatic ketogenesis will be measured using a constant infusion of [3C β-hydroxybutyrate as previously described (Perry et al. Cell Metabolism 2017). Whole body energy expenditure and the respiratory quotient will be assessed by indirect calorimetry.

Official Title
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The Effect of Glucagon on Rates of Hepatic Mitochondrial Oxidation and Pyruvate Carboxylase Flux in Man Assessed by Positional Isotopomer NMR Tracer Analysis (PINTA)

Conditions
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Healthy Participants

Intervention / Treatment
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* Drug: Glucagon


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Healthy Non smoking Taking no medications except birth control T2D fasting plasma glucose 126 - 200 mg/dL, HbA1c >9%, taking metformin, sulfonylureas, DPP4 inhibitors or SGLT2 inhibitors or diet controlled Exclusion Criteria: Any systemic or organ disease except for NAFLD/NASH and T2D T2D taking insulin Smoking Healthy individuals taking any drug or medications other than birth control (women)

Ages Eligible for Study
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Minimum Age: 21 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Intervention Model: Single Group Assignment
Interventional Model Description: Each participant will participate in two studies: one without and one with a 3 hour infusion of glucagon during the PINTA study
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Glucagon<br>Participants will receive glucagon or saline during the PINTA study | Drug: Glucagon<br>* PINTA study with or without glucagon<br>* Other names: hormone study;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Rates of Hepatic Glucose production | Rates of fasting glucose production will be measured using D7 glucose | 5 Hours | 
| Rates of Hepatic Mitochondrial Oxidation | Rates of pyruvate carboxylase flux and citrate synthesis flux will be assessed using GC/MS and NMR analyses of plasma glucose 13C enrichments after the [3-13C]lactate infusion | 5 hours | 
| Rates of Hepatic Ketogenesis | Assessment of hepatic acetyl CoA content will be estimated from rates of hepatic ketogenesis following the 13C beta-hydroxybutyrate infusion | 5 hours | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Hepatic Glucose Metabolism, Hepatic Mitochondrial Oxidation, PINTA, Glucagon