Development and Evaluation of a Glucagon Sensitivity Test in Individuals With and Without Hepatic Steatosis

Glucagon is secreted from pancreatic alpha-cells in response to protein-rich meals and during hypoglycemia. A physiological feedback system exists between the liver and the pancreatic alpha cells termed the liver-alpha cell axis and signifies the role between amino acid-stimulated glucagon secretion and glucagon-stimulated amino acid metabolism.

Individuals with non-alcoholic fatty liver disease have increased levels of glucagon (hyperglucagonemia) and amino acids (hyperaminoacidemia), which suggests that hepatic steatosis may uncouple glucagon's effect on amino acid metabolism (i.e. reduced glucagon sensitivity). Since hyperglucagonemia contributes to diabetes progression - due to its potentiating effects on hepatic glucose production - hepatic steatosis may create a diabetogenic circle. This study aims to develop and evaluate a test for measuring glucagon sensitivity in humans.

The investigators (Associate Prof. Nicolai J Wewer Albrechtsen and Prof. Jørgen Rungby) will investigate whether amino acid metabolism is attenuated in individuals with hepatic steatosis (assessed by magnetic resonance imaging) due to impaired hepatic glucagon sensitivity and if glucagon's effect on hepatic glucose production is intact compared to individuals without hepatic steatosis suggestive of biased signaling.

Amino acids administered orally or intravenously stimulate glucagon secretion from the pancreas and in turn, glucagon is a powerful stimulus for hepatic amino acid turnover through transcriptional (long-term) and non- transcriptional (acute) mechanisms. Several groups including the investigators have linked glucagon secretion to hepatic amino acid metabolism suggesting a mutual feedback cycle, termed the liver-alpha cell axis. A disruption of this axis, which has been shown both pharmacologically using glucagon receptor antagonists and genetically in glucagon receptor knockout mouse models, leads to increased glucagon (hyperglucagonemia) and amino acid (hyperaminoacidemia) concentrations. This phenotype is also evident in subjects with biopsy-verified metabolic dysfunction-associated steatotic liver disease (MASLD) independent of type 2 diabetes suggesting reduced hepatic glucagon sensitivity in the presence of hepatic steatosis. Glucagon increases amino acid catabolism by potentiating ureagenesis, a pathway exclusive to the liver, and hepatic accumulation of triglycerides may reduce glucagon's ability to augment amino acid turnover. Some amino acids are more potent in stimulating glucagon secretion (glucagonotropic amino acids), including alanine, and the glucagon-alanine index is currently used as a surrogate marker for the hepatic actions of glucagon on ureagenesis. The fate of amino acid turnover depends on both glucagon and insulin dynamics by stimulating amino acid catabolism and synthesis, respectively. Studying the effects of glucagon in individuals with type 1 diabetes will allow one to differentiate between the combined effects of glucagon and insulin compared to the effects of glucagon alone.

This study aims to explore hepatic glucagon sensitivity towards amino acid metabolism in individuals with and without hepatic steatosis (based on magnetic resonance imaging (MRI)).

The investigators hypothesize that the effect of endogenous and exogenous glucagon on plasma amino acid levels are impaired in individuals with MASLD (based on hepatic steatosis measured by MRI) compared to controls.

The nomenclature for MASLD has recently been updated from non-alcoholic fatty liver disease (NAFLD) (https://pubmed.ncbi.nlm.nih.gov/37363821/).

Eligibility Criteria:
Group 1 (lean controls)

Inclusion Criteria:

* BMI = 18.6-25 kg/m2
* Male or female
* 25-65 years of age

Exclusion Criteria:

* Diabetes (ADA criteria)
* Significant alcohol/drug abuse as per investigators judgement
* Amino acid-related diseases such as phenylketonuria
* Kidney disease
* Cardiac problems
* Cancer within the past 1 year
* Severe claustrophobia
* Pacemaker or other non-MR-compatible devices
* Pregnancy or breastfeeding.
* Fib4 score \> 3.25.
* Any medicine, acute illness (within the last two weeks) or other circumstances that in the opinion of the investigator might endanger the participants' safety or compliance with the protocol

Group 2 (overweight and obese individuals)

Inclusion Criteria:

* BMI = 25-40 kg/m2
* Male or female
* 25-65 years of age

Exclusion Criteria:

* Diabetes (ADA criteria)
* Significant alcohol/drug abuse as per investigators judgement
* Amino acid-related diseases such as phenylketonuria
* Kidney disease
* Cardiac problems
* Cancer within the past 1 year
* Severe claustrophobia
* Pacemaker or other non-MR-compatible devices
* Pregnancy or breastfeeding
* Abdominal diameter \>70 cm
* Fib4 score \> 3.25.
* Any medicine, acute illness (within the last two weeks) or other circumstances that in the opinion of the investigator might endanger the participants' safety or compliance with the protocol

Group 3 (individuals with type 1 diabetes)

Inclusion Criteria:

* BMI = 18.6-40 kg/m2
* Male or female
* C-peptide negative
* Insulin pump user
* 25-65 years of age

Exclusion Criteria:

* Type 2 diabetes
* Closed loop user
* Microalbuminuria (30-300 mg/g)
* Significant alcohol/drug abuse as per investigators judgement
* Amino acid-related diseases such as phenylketonuria
* Kidney disease
* Cardiac problems
* Cancer within the past 1 year
* Severe claustrophobia
* Pacemaker or other non-MR-compatible devices
* Pregnancy or breastfeeding
* Abdominal diameter \>70 cm
* Fib4 score \> 3.25.
* Any medicine, acute illness (within the last two weeks) or other circumstances that in the opinion of the investigator might endanger the participants' safety or compliance with the protocol

Following inclusion and study completion, our two groups of lean individuals (n=20) and overweight and obese individuals (n=30) will be stratified into two groups based on liver fat content assessed by the magnetic resonance imaging scan. Individuals with \<5.6% hepatic steatosis will be allocated to the control group and individuals with ≥5.6% hepatic steatosis to the MASLD group.