(-)-Epicatechin rich cocoa mediated modulation of oxidative stress regulators in skeletal muscle of heart failure and type 2 diabetes patients

Abstract

Background: Type 2 diabetes (T2D) and heart failure (HF) are associated with high levels of skeletal muscle (SkM) oxidative stress (OS). Health benefits attributed to flavonoids have been ascribed to antioxidation. However, for flavonoids with similar antioxidant potential, end-biological effects vary widely suggesting other mechanistic venues for reducing OS. Decreases in OS may follow the modulation of key regulatory pathways including antioxidant levels (e.g. glutathione) and enzymes such as mitochondrial superoxide dismutase (SOD2) and catalase.

Methods: We examined OS-related alterations in SkM in T2D/HF patients (as compared vs. healthy controls) and evaluated the effects of three-month treatment with (-)-epicatechin (Epi) rich cocoa (ERC). To evidence Epi as the mediator of the improved OS profile we examined the effects of pure Epi (vs. water) on SkM OS regulatory systems in a mouse model of insulin resistance and contrasted results vs. normal mice.

Results: There were severe alterations in OS regulatory systems in T2D/HF SkM as compared with healthy controls. Treatment with ERC induced recovery in glutathione levels and decreases in the nitrotyrosilation and carbonylation of proteins. With treatment, key transcriptional factors translocate into the nucleus leading to increases in SOD2 and catalase protein expression and activity levels. In insulin resistant mice, there were alterations in muscle OS and pure Epi replicated the beneficial effects of ERC found in humans.

Conclusions: Major perturbations in SkM OS can be reversed with ERC in T2D/HF patients. Epi likely mediates such effects and may provide an effective means to treat conditions associated with tissue OS.

Keywords: (−)-epicatechin; (−)-epicatechin rich cocoa; 2,4-dinitrophenyldrazone; Cocoa; DNP; ERC; Epi; Epicatechin; FOXO1; Flavanols; GAPDH; HF; HFD; IP; OS; PGC1α; ROS; S6 ribosomal protein; S6RP; SIRT; SOD2; SkM; T2D; WB; forkhead box protein O1; glyceraldehyde 3-phosphate dehydrogenase; heart failure; high fat diet; immunoprecipitation; oxidative stress; peroxisome proliferator-activated receptor gamma coactivator 1-α; reactive oxygen species; sirtuin; skeletal muscle; superoxide dismutase-2; type 2 diabetes; western blotting.

© 2013.

Figures

Figure 1. Summary of molecular events that participate in the regulation of tissue oxidative stress traced in human and/or mouse SkM samples

This figure annotates the system and experimental approach pursued in documenting tissue oxidative stress related events in skeletal muscle. PCR = polymerase chain reaction, GSH = reduced glutathione, DB = dot blot, IP = immunoprecipitation, WB = Western blot, DNP = dinitrophenyl, DNPH = dinitrophenyl hydrazine, AC = acetyl, P = phospho, ChIP = chromatin immunoprecipitation.

Figure 2. Modulation of SkM oxidative stress markers by ERC in T2D/HF patients before and after treatment

(A) Changes in glutathione levels vs. control. (B) Summary of changes observed in carbonylation (DNP) and nitrotyrosine residue formation (NO2Tyr) vs. control. (Control n=3, patients n=4, *p<0.05 vs. before, # vs. control).

Figure 3. Modulation of SkM SOD2 and SIRT3 by ERC in T2D/HF patients before (B) and after (A) treatment

(A) Changes detected in SIRT3 protein levels as observed by Westerns (normalized with S6RP levels). (B, upper panel) Changes in total SOD2 levels as observed by Westerns and, (C) their quantification. (B, bottom panel) Changes in SIRT3 protein levels following SOD2 based IP and their complexing to SOD2. (D) Decreases observed in SOD2 acetylation and, (E) nitrotyrosine residue formation. (F) Changes observed in SOD2 activity levels vs. control. (Control n=3, patients n=5, *p<0.05 vs. before, # vs. control).

Figure 4. Modulation of SkM catalase by ERC in T2D/HF patients before (B) and after (A) treatment

(A, upper panel) Changes observed in total catalase protein levels and, (B) their quantification. (A, bottom panel) Following catalase based IP, changes in nitrotyrosine (NO2Tyr) residue formation. (C) Changes in the ratio of nitrotyrosilation/total catalase levels. (D) Changes observed in catalase activity levels vs. control. (Control n=3, patients n=5, *p<0.05 vs. before, # vs. control).

Figure 5. Modulation of nuclear events associated with SOD2 and catalase by ERC before (B) and after (A) treatment

(A) Western blot images illustrating the purification of nuclear vs. cytoplasmic material as judged from the blotting with a TATA binding protein (TBP) or GAPDH antibodies. (B) Secondary to IP with an SIRT1 antibody, changes in SIRT1, phospho-SIRT1, FOXO1, acetylated (Ac) FOXO1, PGC1α, Ac-PGC1α. (C) Changes observed in the ratio of phospho-SIRT1 to total SIRT1 protein levels. (D) Changes observed in the ratio of acetylated/total FOXO1 levels. (E) Changes observed in the ratio of acetylated/total PGC1α levels. (n=5, *p<0.05 vs. before).

Figure 6. Modulation of nuclear events associated with SOD2 and catalase promoters by ERC before (B) and after (A) treatment

(A) Changes observed in PGC1α, acetylated (Ac) PGC1α FOXO1 and Ac-FOXO1 levels following chromatin IP with a PGC1α antibody. (B) Changes observed in chromatin associated PGC1α levels. (C) Changes observed in chromatin associated FOXO1 (n=5, *p<0.05 vs. before). (D) PCR generated evidence as to the binding of the above stated transcription factors to the SOD2 and catalase promoters as documented by gel electrophoresis (n=4).

Figure 7. Modulation of SkM oxidative stress regulatory systems by Epi in 6 month old normal and HFD mice with Epi treatment

(A) Changes observed in glutathione levels. (B) Changes observed in total protein carbonylation levels. (C) Differences observed in protein nitrotyrosine (NO2Tyr) residue formation. (n=6/group, *p<0.05 vs. water control, # vs. water-HFD).

Figure 8. Modulation of SkM SOD2 and catalase in 6 month old normal and HFD mice with Epi treatment

(A) Differences observed in acetylated/total SOD2 protein levels. (B) Changes observed in SOD2 activity. (C) Differences observed in catalase nitrotyrosine residue (NO2Tyr) formation. (D) Differences observed in catalase activity levels. (n=6/group, *p<0.05 vs. water control, # vs. water-HFD).