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Objectives: Experimental and genetic studies suggest that fibroblast growth factor 21 (FGF21) modulates macronutrient and alcohol preferences, but evidence of such regulation in humans remains scarce. To address this gap in translation, we aimed to map the relationships between plasma FGF21 levels, FGF21 genetic variation and habitual macronutrient intake in a large human population. Methods: We fine-mapped and performed colocalization of the FGF21 genetic region in GWAS summary statistics of plasma FGF21 levels and macronutrient intake. UK Biobank data were used to investigate the associations between FGF21 genetic variants, plasma FGF21 protein levels, and macronutrient intake (including alcohol) assessed with repeated 24-hour recalls. One- and two-sample mendelian randomization were performed to estimate the effects of plasma FGF21 on macronutrient intake. Results: We show that the main macronutrient-associated variant rs838133 and the FGF21 cis-pQTL rs838131, both in the FGF21 gene, are distinct genetic signals. Effect directions also suggest that the influence of FGF21 variation on macronutrient intake appear more complex than by direct mediation through plasma FGF21. Only when considering this complexity at FGF21, is plasma FGF21 estimated to reduce alcohol and increase protein and fat intake using mendelian randomization. Importantly, plasma FGF21 levels also appear markedly elevated by primarily high alcohol and low protein intake. Conclusions: These findings support the feedback diet-regulatory mechanism of FGF21 in humans, but highlights the need for mechanistic characterization of the complex FGF21 genetic region.

N-acyl taurines (NATs) are bioactive lipids with emerging roles in glucose homeostasis and lipid metabolism. The acyl chains of hepatic and biliary NATs are enriched in polyunsaturated fatty acids (PUFAs). Dietary supplementation with a class of PUFAs, the omega-3 fatty acids, increases their cognate NATs in mice and humans. However, the synthesis pathway of the PUFA-containing NATs remains undiscovered. Here, we report that human livers synthesize NATs and that the acyl-chain preference is similar in murine liver homogenates. In the mouse, we found that hepatic NAT synthase activity localizes to the peroxisome and depends upon an active-site cysteine. Using unbiased metabolomics and proteomics, we identified bile acid-CoA:amino acid N-acyltransferase (BAAT) as the likely hepatic NAT synthase in vitro. Subsequently, we confirmed that BAAT knockout livers lack up to 90% of NAT synthase activity and that biliary PUFA-containing NATs are significantly reduced compared with wildtype. In conclusion, we identified the in vivo PUFA-NAT synthase in the murine liver and expanded the known substrates of the bile acid-conjugating enzyme, BAAT, beyond classic bile acids to the synthesis of a novel class of bioactive lipids.

Objective: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. Methods: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). Results: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). Conclusions: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.

Objective: Excessive alcohol consumption is a leading cause of global morbidity and mortality. However, knowledge of the biological factors that influence ad libitum alcohol intake may be incomplete. Two large studies recently linked variants in the KLB locus with levels of alcohol intake in humans. KLB encodes β-klotho, co-receptor for the liver-derived hormone fibroblast growth factor 21 (FGF21). In mice, FGF21 reduces alcohol intake, and human Fgf21 variants are enriched among heavy drinkers. Thus, the liver may limit alcohol consumption by secreting FGF21. However, whether full-length, active plasma FGF21 (FGF21 (1–181)) levels in humans increase acutely or sub-chronically in response to alcohol ingestion is uncertain. Methods: We recruited 10 healthy, fasted male subjects to receive an oral water or alcohol bolus with concurrent blood sampling for FGF21 (1–181) measurement in plasma. In addition, we measured circulating FGF21 (1–181) levels, liver stiffness, triglyceride, and other metabolic parameters in three healthy Danish men before and after consuming an average of 22.6 beers/person/day (4.4 g/kg/day of ethanol) for three days during Oktoberfest 2017 in Munich, Germany. We further correlated fasting FGF21 (1–181) levels in 49 healthy, non-alcoholic subjects of mixed sex with self-reports of alcohol-related behaviors, emotional responses, and problems. Finally, we characterized the effect of recombinant human FGF21 injection on ad libitum alcohol intake in mice. Results: We show that alcohol ingestion (25.3 g or ∼2.5 standard drinks) acutely increases plasma levels of FGF21 (1–181) 3.4-fold in fasting humans. We also find that binge drinking for three days at Oktoberfest is associated with a 2.1-fold increase in baseline FGF21 (1–181) levels, in contrast to minor deteriorations in metabolic and hepatic biomarkers. However, basal FGF21 (1–181) levels were not correlated with differences in alcohol-related behaviors, emotional responses, or problems in our non-alcoholic subjects. Finally, we show that once-daily injection of recombinant human FGF21 reduces ad libitum alcohol intake by 21% in mice. Conclusions: FGF21 (1–181) is markedly increased in circulation by both acute and sub-chronic alcohol intake in humans, and reduces alcohol intake in mice. These observations are consistent with a role for FGF21 as an endocrine inhibitor of alcohol appetite in humans. Keywords: Fibroblast growth factor 21, FGF21, Alcohol, Alcohol appetite

The unfolded protein response (UPR), induced by endoplasmic reticulum (ER) stress, regulates the expression of factors that restore protein folding homeostasis. However, in the liver and kidney, ER stress also leads to lipid accumulation, accompanied at least in the liver by transcriptional suppression of metabolic genes. The mechanisms of this accumulation, including which pathways contribute to the phenotype in each organ, are unclear. We combined gene expression profiling, biochemical assays, and untargeted lipidomics to understand the basis of stress-dependent lipid accumulation, taking advantage of enhanced hepatic and renal steatosis in mice lacking the ER stress sensor ATF6α. We found that impaired fatty acid oxidation contributed to the early development of steatosis in the liver but not the kidney, while anorexia-induced lipolysis promoted late triglyceride and free fatty acid accumulation in both organs. These findings provide evidence for both direct and indirect regulation of peripheral metabolism by ER stress.