State Anxiety Is Associated with Cardiovascular Reactivity in Young, Healthy African Americans

Although several studies have shown that enhanced cardiovascular reactivity can predict hypertension development in African Americans, these findings have not been consistent among all studies examining reactivity and hypertension susceptibility. This inconsistency may be explained by the influence of anxiety (state and trait) on the blood pressure response to stress. Therefore, this study sought to determine whether anxiety is associated with blood pressure response to cold pressor (CP) and anger recall (AR) stress tests in young healthy African Americans. Modeling using state and trait anxiety revealed that state anxiety predicts systolic (SBP) and diastolic blood pressure DBP response to CP and AR (P ≤ 0.02). Interestingly, state anxiety predicted heart rate changes only to CP (P < 0.01; P = 0.3 for AR). Although trait anxiety was associated with SBP response to AR and not CP, it was not a significant predictor of reactivity in our models. We conclude that anxiety levels may contribute to the variable blood pressure response to acute stressors and, therefore, should be assessed when performing cardiovascular reactivity measures

Effects of leucine supplementation and serum withdrawal on branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability

Effect of different treatments on expression levels of BCAA metabolism pathway genes.

<p>Quantitative PCR (qPCR) data was generated from three independent cell cultures for each treatment-timepoint combination and analyzed in triplicate. Gene abundances were normalized to <i>Gapdh</i> levels and then multiplied by a factor of 10,000 to avoid very small values. The relative abundance measures for each of the replicate cell cultures were combined to provide a summary abundance measure. Panels, from top to bottom – genes common to the metabolism of all 3 BCAAs; genes specific to valine metabolism; genes specific to leucine metabolism; genes specific to isoleucine metabolism. Gene symbols are indicated at the top of each plot. Treatments are shown as C (control, open rectangle), L (leucine, hatched rectangle), and SW (serum withdrawal, black rectangle). Results are shown for each of Day0, Day4 and Day10. <i>GOI</i>, gene of interest.</p

Effects of leucine supplementation and serum withdrawal on 3T3-L1 adipocyte differentiation.

<p><b>a.</b> Oil-red O staining of cell cultures at Day 0, Day 4 and Day 10 under <i>control, leucine or serum withdrawal</i> conditions. Cells were grown under the defined conditions for the specified periods of time and stained for total neutral lipids. <b>b.</b> Quantitative PCR analysis of key genes associated with adipocyte differentiation. Gene names are indicated on the plots. Gene expression abundances were estimated by the ΔΔQ method after normalizing to GAPDH and referencing to Control, Day 0 samples. Due to the relatively high range of fold-change for adiponectin and CEBP-alpha, their relative abundances are expressed in the log₁₀ scale. The treatments and stages of differentiation are indicated at the bottom (C,<i>control</i>; L, <i>leucine</i>; SW, <i>serum-withdrawal</i>).</p

Western blot analysis of Bcat2 and Bckdha protein levels in treated cells over the course of adipocyte differentiation.

<p><b>a.</b> Protein expression from three independent cell-culture experiments is shown for each gene. Treatments (<i>control, leucine or serum withdrawal</i>) are indicated at the left of each blot and the timepoints are indicated at the top. Mitogen activated protein kinase (Mapk) was used as loading control. <b>b.</b> Quantification of relative protein abundances by scanning densitometry on Western blots. Protein abundances are expressed relative to Mapk levels and average values from three independent cell-culture studies are shown. Significant changes in protein expression (p<0.05) are indicated as follows – comparison vs. Day 0, open squares; comparison against <i>serum withdrawal</i>, open star; comparison of Day 10 vs. Day 4, open circle; comparison of <i>leucine</i> vs. <i>control</i>, open triangle.</p