Fig. 1: cAMP dose-responses with different agonists in HEK293 cells.

A Intracellular cAMP production in stably transfected β2-HEK293 cells after treating them with increasing concentration of isoproterenol (ISO) (Blue), norepinephrine (NE) (Red), phenylacetylglutamine (PAGln) (Purple) and phenylalanine (Phe) (Black) as indicated. B Intracellular cAMP production in stably transfected β1-HEK293 cells after treating them with increasing concentration of isoproterenol (ISO) (Blue), norepinephrine (NE) (Red), phenylacetylglutamine (PAGln) (Purple) and phenylalanine (Phe) (Black) as indicated. ISO and NE serve as positive controls and phenylalanine (Phe) serves as negative control. C cAMP dose-response in stably transfected β2-HEK293 cells after treating the cells with isoproterenol (ISO) alone (Blue), or ISO along with 100 µM PAGln (Red), pre-incubated for 15 min (P < 0.0001; EC₅₀ Blue vs Red). For ISO-induced cAMP dose-response, PAGln pre-incubation in β2-HEK293 displayed 5.3 ± 0.80-fold increase (P = 0.04) in the EC₅₀ as compared to the ISO alone control. Two-tailed unpaired Welch’s student t-test was used to obtain the fold change P-value by comparing the mean EC₅₀ values of ISO and ISO+PAGIn from three different experiments (ISO vs ISO+PAGln; P = 0.04. D cAMP dose-response in stably transfected β1-HEK293 cells after treating the cells with isoproterenol (ISO) alone (Blue), or ISO along with 100 µM PAGln (Red), pre-incubated for 15 min (P = 0.75; EC₅₀ Blue vs Red). E cAMP dose-response curves of norepinephrine (NE) alone (Blue) and NE along with 100 µM PAGln (Red), pre-incubated for 15 min in stably transfected β2-HEK293 cells (P < 0.0001; EC₅₀ Blue vs Red). PAGln pre-incubation in β2-HEK293 displays 4.2 ± 1.69-fold increase (P = 0.04) in the EC₅₀ of the dose-response of NE pre-incubated with PAGln as compared to NE alone control. Two-tailed unpaired Welch’s student t-test was used to obtain the fold change P-value when comparing the EC₅₀ values for NE and NE+PAGIn from three different experiments (NEvs NE+PAGIn; P = 0.04). F cAMP dose-response curves of norepinephrine (NE) alone (Blue) and NE along with 100 µM PAGln (Red), pre-incubated for 15 min in stably transfected β1-HEK293 cells (P = 0.47; EC₅₀ Blue vs Red). Data points represent the mean ± SD of the indicated number (n) of experiments. P values (top left) for EC₅₀ comparisons (Blue vs Red) as well as the standard error of the mean EC₅₀ (SEM) were determined with a non-linear regression method and the significance of the fold changes was analyzed using two-tailed unpaired Welch’s student t-test. Nonparametric two-tailed Mann–Whitney test was used for non-pairwise comparisons (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). All reported P values are two-sided. A P-value of <0.05 was considered significant in this study.