Wednesday, April 2, 2025
4/2/2025
CYP4F Enzymes Regulate N-Acyl Amino Acid Signaling in Humans - PROJECT SUMMARY Obesity and obesity-related type 2 diabetes (T2D) pose major health threats in the US, however the early pathophysiology of these diseases remain incompletely defined. Blood-borne lipid signaling molecules called lipokines are an emerging class of endocrine factors that control systemic energy and glucose homeostasis and offer significant potential as therapeutic targets in human metabolic disease. We recently discovered that an unusual family of lipokines that consist of fatty acid-amino acid conjugates called N-acyl amino acids stimulate energy expenditure and thereby reduce body weight and improve glucose tolerance in murine models of obesity. To define the role of N-acyl amino acids in human biology, we recently used mass spectrometry (MS) methods to measure N-acyl amino acid family members in 2,351 participants of the Jackson Heart Study (JHS) and found that individual N-acyl amino acids are strongly associated with decreased risk of future T2D, coronary heart disease, and heart failure. To identify the genetic determinants of these disease-associated N-acyl amino acids in humans, we performed genome-wide association studies on plasma levels of each N-acyl amino acid in JHS and unexpectedly identified a strong association between the CYP4F gene locus and specific N-acyl amino acids. We experimentally validated this association by demonstrating that CYP4F2 directly hydroxylates specific N-acyl amino acids using cell models. Moreover, we demonstrated that this reaction competitively inhibits the hydroxylation of CYP4F2 canonical enzymatic substrates such as arachidonic acid and other key eicosanoids, and produces numerous previously unknown lipid metabolites, including several that structurally resemble anti-diabetic fatty acid hydroxy fatty acids (FAHFAs). Based on these findings, we hypothesize that N-acyl-amino acids serve as endogenous substrates of CYP4F enzymes in humans and that this biochemical interaction has important functional consequences on downstream lipid signaling networks and cellular metabolic function. In Specific Aim 1, we will biochemically characterize N-acyl amino acids as substrates and competitive inhibitors of CYP4F enzymes using cultured primary human hepatocytes and recombinant CYP4F proteins. In Specific Aim 2, we will define the functional consequences of N-acyl amino acid inactivation by CYP4F hydroxylation on cellular metabolism using cultured primary human hepatocytes. In Specific Aim 3, we will characterize novel N-acyl amino acid downstream products of CYP4F hydroxylation as biomarkers of cardiometabolic disease in two, diverse, large human population studies. Successful completion of this project will establish a biochemical basis for the association of the CYP4F gene locus with N-acyl amino acids in humans and will serve as a critical first step toward understanding the physiologic functions of N-acyl amino acids in human metabolism. Characterization of N- acyl amino acids and related pathways may ultimately provide new insight into therapeutic strategies for the treatment and prevention of obesity and T2D.
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