Correlation of the change of plasma cysteinylglycine and hexadecanedioate levels with the antihypertensive effect of amlodipine.

<p>Correlation coefficients (r) and P values from partial correlation, calculated with normalized metabolite change values and controlling for metabolite baseline level, are included. dASBP, change of 24-hour ambulatory systolic blood pressure; dADBP, change of 24-hour ambulatory diastolic blood pressure.</p

Characteristics of the study subjects.

<p>Characteristics of the study subjects.</p

Effect of antihypertensive drugs on selected plasma long-chain fatty acids.

<p>Plasma metabolite level is presented as relative units: the median of all analyzed samples was set to 1. Box-and-whisker plots are presented. P values <0.05 from Wilcoxon signed-rank test are included. P, placebo (mean of three periods); A, amlodipine; B, bisoprolol; H, hydrochlorothiazide; L, losartan.</p

Effect of antihypertensive drugs on selected plasma acylcarnitines.

<p>Plasma metabolite level is presented as relative units: the median of all analyzed samples was set to 1. Box-and-whisker plots are presented. P values <0.05 from Wilcoxon signed-rank test are included. P, placebo (mean of three periods); A, amlodipine; B, bisoprolol; H, hydrochlorothiazide; L, losartan.</p

Amino acid metabolites that regulate G protein signaling during osmotic stress

<div><p>All cells respond to osmotic stress by implementing molecular signaling events to protect the organism. Failure to properly adapt can lead to pathologies such as hypertension and ischemia-reperfusion injury. Mitogen-activated protein kinases (MAPKs) are activated in response to osmotic stress, as well as by signals acting through G protein-coupled receptors (GPCRs). For proper adaptation, the action of these kinases must be coordinated. To identify second messengers of stress adaptation, we conducted a mass spectrometry-based global metabolomics profiling analysis, quantifying nearly 300 metabolites in the yeast <i>S</i>. <i>cerevisiae</i>. We show that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the MAPK Hog1. Ectopic addition of these BCAA derivatives promotes phosphorylation of the G protein α subunit and dampens G protein-dependent transcription, similar to that seen in response to osmotic stress. Conversely, genetic ablation of Hog1 activity or the BCAA-regulatory enzymes leads to diminished phosphorylation of Gα and increased transcription. Taken together, our results define a new class of candidate second messengers that mediate cross talk between osmotic stress and GPCR signaling pathways.</p></div

BCAA derivatives diminish MAPK-dependent gene transcription.

<p>(A) Addition of BCAA derivatives or KCl dampens α-factor pheromone-induced gene transcription (P_FUS1-GFP). The dampening capacity of each BCAA derivative is correlated with Gpa1 phosphorylation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006829#pgen.1006829.g004" target="_blank">Fig 4</a>). (B) Genetic ablation of the AMPK kinases increases basal gene transcription, consistent with reduced Gpa1 phosphorylation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006829#pgen.1006829.ref021" target="_blank">21</a>]. Correspondingly, the kinase mutants abrogate any ability of the metabolites to suppress basal signaling and limit their ability to suppress pheromone signaling (41% reduction in wildtype vs. 26% reduction in the kinase mutant strain). Data are presented as mean ± standard deviation, N = 4.</p

BCAA derivatives promote Gpa1 phosphorylation.

<p>(A) Ectopic addition of 2-hydroxyisocaproate (HIC) promotes phosphorylation of Gpa1 but not Hog1 or Snf1. (B) Ectopic addition of the BCAA derivatives promotes Gpa1 phosphorylation while intracellular pH is unaffected (inset). (C, D) Ectopic addition of HIC promotes Gpa1 phosphorylation in wild-type and Hog1-deficient cells. Osmotic stress promotes Gpa1 phosphorylation only in wild-type cells. Data presented as mean ± standard deviation, N = 3.</p

Phosphorylation of Gpa1 in response to osmotic stress occurs in a Hog1-dependent, pH-independent manner.

<p>Western blot analysis reveals that Gpa1 and Snf1 are phosphorylated (p-Gpa1 and p-Snf1) in response to (A) glucose (Glc) limitation (“High” = 2% glucose, “Low” = 0.05% glucose), (B) heat shock (42°C), or (C) osmotic stress (0.5 M KCl). Note that Hog1 is phosphorylated (p-Hog1) in response to heat shock or osmotic stress, but not glucose limitation. Intracellular pH (insets) decreases in response to glucose limitation or heat shock (shaded area), but not osmotic stress. Hog1 catalytic activity (<i>hog1</i>^<i>K52R</i>) is required for phosphorylation of Gpa1 but not Snf1. Diploid, control cells lacking Gpa1. <i>reg1</i>Δ, control cells lacking Gpa1 phosphatase. Hog1, Snf1, and Load correspond to gels probed with Hog1, poly-His, and G6PDH antibodies, respectively. Data were quantified based on band intensity, and are presented as mean ± standard deviation, N = 3.</p

BCAA derivatives do not bind directly to Gα proteins.

<p>¹H-¹⁵N 2D HSQC NMR spectra of Gαi-GDP alone (black) or in the presence of 25-fold excess (A) HIV, (B) HIC, or (C) HMVA (color) reveal no discernable peak shifts. (D) Spectral overlay of Gαi-GDP at pH 6.0 (magenta) and pH 7.0 (black) is presented as a positive control. Inset, magnified view of a subset of resonances showing pH-dependent spectral changes.</p

The AMPK kinase Elm1 phosphorylates Gpa1 upon BCAA derivative addition.

<p>Gpa1 phosphorylation after ectopic addition of 30 mM HIC is abrogated in cells lacking the AMPK kinase <i>ELM1</i>, or all three AMPK kinases (ΔΔΔ). Data presented as mean ± standard deviation, N = 3.</p