A Single Oral Administration of Theaflavins Increases Energy Expenditure and the Expression of Metabolic Genes

<div><p>Theaflavins are polyphenols found in black tea, whose physiological activities are not well understood. This study on mice evaluated the influence of a single oral administration of theaflavins on energy metabolism by monitoring the initial metabolic changess in skeletal muscle and brown adipose tissue (BAT). Oxygen consumption (VO₂) and energy expenditure (EE) were increased significantly in mice treated with theaflavin rich fraction (TF) compared with the group administered vehicle alone. There was no difference in locomotor activity. Fasting mice were euthanized under anesthesia before and 2 and 5, 20-hr after treatment with TF or vehicle. The mRNA levels of uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in BAT were increased significantly 2-hr after administration ofTF. The levels of UCP-3 and PGC-1α in the gastrocnemius muscle were increased significantly 2 and 5-hr after administration of TF. The concentration of phosphorylated AMP-activated protein kinase (AMPK) 1α was also increased significantly in the gastrocnemius 2 and 5-hr after treatment with TF. These results indicate that TF significantly enhances systemic energy expenditure, as evidenced by an increase in expression of metabolic genes.</p></div

Expression of mRNA for UCP-1 in BAT (a) and UCP-3 in gastrocnemius muscle (b) after administration of vehicle or theaflavin.

<p>The animals were euthanized before and 2, 5, and 20 hr after administration of vehicle or 10 mg/kg theaflavin (n = 8). The values represent the mean ± standard deviation. The statistical analyses were performed two way ANCOVA post hoc comparisons with the vehicle group were made by the two-tailed followed by Dunnett's test. Significantly different from vehicle, *p<0.05, **p<0.01.</p

Expression of mRNA for PGC1-α in BAT (a) or gastrocnemius (b) after administration of vehicle or theaflavin.

<p>The animals were euthanized before and 2, 5, and 20 hr after administration of vehicle (n = 8) or 10 mg/kg theaflavin (n = 8). The values represent the mean ± standard deviation. The statistical analyses were performed two way ANCOVA, post hoc comparisons with the vehicle group were made by the two-tailed followed by Dunnett's test. Significantly different from vehicle, *p<0.05.</p

Oxygen consumption (VO₂₎ and energy expenditure (EE) 20 hr after administration of theaflavin.

<p>The respiratory exchange ratio (RER) was calculated using VO₂ and carbon dioxide excretion (VCO₂) and the Weir equation. Total VO₂ and EE during the light (12:00–18:00 and 6:00–8:00) or dark (18:00–6:00) cycles are shown in b and d. The locomotor activity of the animals 20 hr after administration of vehicle or theaflavin is shown in e, while locomotor activity of the mice during the total, light or dark cycles is shown in f. The mice were administrated either vehicle (n = 8) or 10 mg/kg theaflavin (n = 8). The values represent the mean ± standard deviation. The statistical analyses were performed two way ANCOVA (a,c,e) post hoc comparisons with the vehicle group were made by the two-tailed followed by Dunnett's test. Significantly different from vehicle, *p<0.05.</p

Comparison of the sympathetic stimulatory abilities of B-type procyanidins based on induction of uncoupling protein-1 in brown adipose tissue (BAT) and increased plasma catecholamine (CA) in mice

<div><p>Objectives</p><p>We previously found that elevated energy expenditure following a single oral dose of flavan 3-ols (FL), a mixture of catechins and B type procyanidins, is caused by sympathetic nerve activation. In the present study, we compared the activity of the FL components (-)-epicatechin (EC; monomer), procyanidin B2 (B2; dimer), procyanidin C1 (C1; trimer), cinnamtannin A2 (A2; tetramer), and more than pentamer fraction (P5).</p><p>Methods</p><p>Male ICR mice were treated with a single oral dose of FL, EC, B2, C1, A2, or P5. The animals were sacrificed and blood and brown adipose tissue (BAT) sampled. The plasma catecholamine (CA) levels and BAT uncoupling protein (UCP)-1 mRNA expression were determined.</p><p>Results</p><p>A single dose of 10 mg/kg FL significantly increased plasma CA and UCP-1 mRNA levels. B2, C1, and A2, but not EC and P5 (all at 1 mg/kg), significantly increased plasma adrenaline levels. Plasma noradrenaline was significantly elevated by B2 and A2, but not by EC, C1, or P5. UCP-1 mRNA levels were significantly increased by C1 and P5. In the dose response study of A2, 10⁻³ mg/kg A2 increased UCP-1 mRNA levels significantly, but not 10⁻² and 10⁻¹ mg/kg A2. In addition, combination treatment with 10⁻¹ mg/kg A2 and yohimbine, an α2 adrenalin blocker, remarkably increased UCP-1 mRNA levels.</p><p>Conclusion</p><p>These results suggest that FL and its components, except EC, increase UCP-1 mRNA and plasma CA with varying efficacy.</p></div

Structures of B type procyanidin.

<p>(a) (-)-epicatechin (EC; monomer), (b) procyanidin B2 (B2; dimer), (c) procyanidin C1 (C1; trimer), (d) cinnamtannin A2 (A2; tetramer).</p

Alterations in plasma noradrenaline (a), adrenaline (b), and UCP-1 mRNA in BAT (c) 2 h after ingestion of 1 mg/kg EC, B2, C1, A2, P5, or vehicle.

<p>The values represent mean ± standard deviation (each group, n = 8). **p<0.01. *p<0.05, **p<0.01 (Tukey-Kramer test vs. vehicle)</p

Dose-reactive response of UCP-1 mRNA levels to A2 and A2 plus yohimbine in BAT.

<p>The values represent mean ± standard deviation (each group, n = 8). *p<0.05, **p<0.01 (Tukey-Kramer test between experimental group).</p

Alterations in plasma noradrenaline (a), adrenaline (b), and UCP-1 mRNA levels in BAT (c) after ingestion of 10 mg/kg flavan 3-ols or vehicle.

<p>The values represent mean ± standard deviation (each group, n = 8). *p<0.05, **p<0.01 (Tukey-Kramer test vs. vehicle).</p

ZER induced HSPs expressions through HSF1 activation.

<p>(A) Hepa1c1c7 cells were treated with ZER (100 µM) for 1 or 3 hours. HSF1 phosphorylated at Ser326 in cell lysates was detected by ELISA. As a positive control, cells were exposed to heat shock (HS; incubation at 43°C in waterbath) for 15 minutes and examined. This experiment was performed in quadruplicates. (B) Cells were treated with the vehicle, ZER (Z; 10, 25, 50 µM) or geldanamycin (G; 1 µM) for 3 hours, then total RNA was subjected to qRT-PCR to semi-quantify the expressions of HSP90α, HSP90β, HSP70, and HSP40. HPRT expressions were also measured as internal standards. This experiment was performed in triplicates. a, versus CTL by Dunnett's test (<i>P</i><0.05). (C) Cells were treated with the vehicle, ZER (50 µM), or geldanamycin (GEL; 1 µM) for 6-24 hours, then lysed for western blot analysis. (D) Cells were treated with Lipofectamine™ 2000 and a siRNA solution (control and HSF1, 75 nM) for 6 hours. The culture medium was replaced with DMEM containing 10% FBS and incubated for another 24 hours. Cell lysates were subjected to western blot analysis. (E) Cells were treated with Lipofectamine™ 2000 and a siRNA solution (control and HSF1, 75 nM) for 6 hours, then the culture medium was replaced with DMEM containing 10% FBS and incubation was performed for another 24 hours. Then, siRNA-transfected cells were treated with the vehicle, ZER (50 µM), or geldanamycin (GEL; 1 µM) for 15 hours, and total RNA was subjected to qRT-PCR to semi-quantify the expression of HSP70. HPRT expression was also determined as an internal standard. This experiment was performed in quadruplicates. The various characteristics were significantly different, as shown by Tukey-Kramer test result (<i>P</i><0.05). (F) Cells were treated with the vehicle, ZER (50 µM) or tunicamycin (TUN; 1 µM) for 6 hours, then total RNA was subjected to qRT-PCR to semi-quantify the expressions of CHOP and GRP78. HPRT expressions were also measured as internal standards. This experiment was performed in triplicates. *<i>P</i><0.05 vs. DMSO by Dunnett's test.</p