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. Author manuscript; available in PMC: 2021 Feb 26.
Published in final edited form as: Sci Transl Med. 2020 Aug 26;12(558):eaaz8664. doi: 10.1126/scitranslmed.aaz8664

Fig. 7. HUMBLE cells display elevated arginine and NO metabolism.

Fig. 7.

(A to C) Principal component analysis (PCA) plot (A), pathway enrichment (B), and clustered heatmap (C) from metabolomics analysis of secreted media from differentiated white control, HUMBLE, or brown control adipocytes. Pathway impact in (B) indicates the degree centrality of changed metabolites in the pathway. Metabolites in (C) in red text are involved in arginine and proline metabolism; N = 6 technical replicates per group. (D and E) Relative abundance of arginine, citrulline, and ornithine in secreted media (D) and cell lysates (E) of differentiated white control, HUMBLE, or brown control adipocytes; N = 6 replicates per group for medium, N=4 replicates per group for lysate. (F) eNOS and ARG2 mRNA expression were measured by qPCR in white control, HUMBLE, and brown control adipocytes; n = 4 biological replicates per group. (G and H) Total NOS activity (G) and total nitric oxide concentration (nitrite and nitrate) in the medium (H) from differentiated white control, HUMBLE, and brown control adipocytes; n = 3 biological replicates per group for NOS activity, 5 biological replicates per group for total nitric oxide concentration. (I) Schematic of arginine metabolism pathway showing genes and metabolites differentially regulated in in vitro differentiated HUMBLE adipocytes compared to white control adipocytes. Strong (red) or slight (pink) increase of genes and metabolites in HUMBLE adipocytes compared to white control adipocytes. NOS, nitric oxide synthase; ARG, arginase; OTC, ornithine transcarbamylase; OAT, ornithine aminotransferase; ASS, argininosuccinate synthase; ASL, argininosuccinate lyase; AGAT, arginine:glycine amidinotransferase; GAMT, guanidinoacetate N-methyltransferase. (J) Glucose uptake in mouse (left) and human (right) brown adipocytes after 24-hr co-culture with white control, HUMBLE, and HUMBLE adipocytes with eNOS KO; N = 3 technical replicates per group. For all charts data are presented as means ± s.e.m.; *p < 0.05; **p < 0.01; ***p < 0.001.