Circadian modulation of the cardiac proteome underpins differential adaptation to morning and evening exercise training: an LC-MS/MS analysis.

All living beings on earth are influenced by the circadian rhythm, the rising and the setting of the sun. The ubiquitous effect of exercise is widely believed to maximize health benefits but has not been formally investigated for cardiac responses in the exercise-induced circadian rhythms. We hypothesized that the exercise-related proteome is differentially influenced by circadian rhythm and analyzed the differences between the effects of morning and evening exercise. Twenty-four Sprague-Dawley rats were randomly divided into four groups (n = 6 per group): morning control, morning exercise, evening control, and evening exercise groups. The exercise groups were subjected to 12-week treadmill exercise (5 days/week) performed either during daytime or nighttime. After 12 weeks, the physiological characteristics (e.g., body weight, heart weight, visceral fat, and blood metabolites), cardiovascular capacity (ejection fraction (%) and fractional shortening (%)), circadian gene expression levels (clock, ball1, per1, per2, cry1, and cry2), and the proteomic data were obtained and subjected to univariate and multivariate analysis. The mRNA levels of per1 and cry2 increased in the evening group compared with those in the morning group. We also found that per2 decreased and cry2 increased in the evening exercise groups. The evening exercise groups showed more decreased triacylglycerides and increased blood insulin levels than the morning exercise group. The principal component analysis, partial least squares discriminant analysis, and orthogonal partial least squares discriminant analysis indicated that the circadian rhythm differently influenced the protein networks of the exercise groups. In the morning exercise group, the transcription-translation feedback loop (TTFL) (clock, per1, per2, cry1, and cry2) formed a protein-protein interaction network with Nme2, Hint1, Ddt, Ndufb8, Ldha, and Eef1a2. In contrast, the TTFL group appeared close to Maoa, Hist2h4, and Macrod1 in the evening exercise group. Interestingly, the evening exercise group decreased the mRNA level of per2 but not per1. Per1 and Per2 are known to transport Cry1 and Cry2 into the nucleus. Taken together, we summarized the characteristics of enriched proteins in the aspect of their molecular function, cellular component, and biological process. Our results might provide a better understanding of the circadian effect on exercise-related proteins.

Spinochrome D Attenuates Doxorubicin-Induced Cardiomyocyte Death via Improving Glutathione Metabolism and Attenuating Oxidative Stress.

Doxorubicin, an anthracycline from Streptomyces peucetius, exhibits antitumor activity against various cancers. However, doxorubicin is cardiotoxic at cumulative doses, causing increases in intracellular reactive oxygen species in the heart. Spinochrome D (SpD) has a structure of 2,3,5,6,8-pentahydroxy-1,4-naphthoquinone and is a structural analogue of well-known sea urchin pigment echinochrome A. We previously reported that echinochrome A is cardioprotective against doxorubicin toxicity. In the present study, we assessed the cardioprotective effects of SpD against doxorubicin and determined the underlying mechanism. ¹H-NMR-based metabolomics and mass spectrometry-based proteomics were utilized to characterize the metabolites and proteins induced by SpD in a human cardiomyocyte cell line (AC16) and human breast cancer cell line (MCF-7). Multivariate analyses identified 12 discriminating metabolites (variable importance in projection > 1.0) and 1814 proteins from SpD-treated AC16 cells. Proteomics and metabolomics analyses showed that glutathione metabolism was significantly influenced by SpD treatment in AC16 cells. SpD treatment increased ATP production and the oxygen consumption rate in D-galactose-treated AC16 cells. SpD protected AC16 cells from doxorubicin cytotoxicity, but it did not affect the anticancer properties. With SpD treatment, the mitochondrial membrane potential and mitochondrial calcium localization were significantly different between cardiomyocytes and cancer cell lines. Our findings suggest that SpD could be cardioprotective against the cytotoxicity of doxorubicin.

Neopetroside-B alleviates doxorubicin-induced cardiotoxicity via mitochondrial protection.

Doxorubicin, a member of the anthracycline family, is a widely prescribed anticancer chemotherapy drug. Unfortunately, cumulative doses of doxorubicin can cause mitochondrial dysfunction, leading to acute or chronic cardiotoxicity. This study demonstrated that Neopetroside-B (NPS-B) protects cardiomyocytes in the presence of doxorubicin. NPS-B improved mitochondrial function in cardiomyocytes by increasing ATP production and oxygen consumption rates. On the other hand, NPS-B negatively influenced cancer cell lines by increasing reactive oxygen species. We analyzed NPS-B-influenced metabolites (VIP > 1.0; AUC>0.7; p < 0.05) and proteins (FC > 2.0) and constructed metabolite-protein enrichment, which showed that NPS-B affected uracil metabolism and NAD-binding proteins (e.g., aldehyde dehydrogenase and glutathione reductase) in cardiomyocytes. However, for the cancer cells, NPS-B decreased the NAD+/NADH balance, impairing cell viability. In a xenograft mouse model treated with doxorubicin, NPS-B reduced cardiac fibrosis and improved cardiac function. NPS-B may be a beneficial intervention to reducing doxorubicin-induced cardiotoxicity with anticancer effects.

Induction of heat shock protein 72 in C6 glioma cells by methyl jasmonate through ROS-dependent heat shock factor 1 activation.

Salicylate and jasmonates are two different types of plant hormone that play critical roles in plant defense responses against insect herbivores and microbial pathogens, through activating defense genes. These two natural products have been shown to have similar activities in animal cells: the compounds are able to induce cell cycle arrest or apoptosis in a variety of human cancer cells including those of colon, prostate, breast, and leukemia, suggesting the chemicals may potentially be a novel class of anti-cancer drugs. Since sodium salicylate can induce the heat shock response in animals, we examined the effects of jasmonates on the heat shock response in C6 glioma cells. Here, we show that brief exposure to methyl jasmonate (MeJA), but not to jasmonic acid, induces heat shock protein 72 (HSP72), but not HSP73 and HSP90, via heat shock factor I (HSF1) activation in C6 glioma cells without affecting cell viability. Intracellular H2O2 and O2-, and mitochondrial ROS were prominently increased in response to 5 mM MeJA in C6 cells. MeJA-induced HSP72 expression, HSF1 DNA binding, and human HSP70 promoter-driven CAT activity were prevented by N-acetyl-L-cysteine (a general antioxidant), catalase (a specific antioxidant for H2O2), and sodium formate (an inhibitor of OH.), but not by Rac1 dominant negative mutant Rac1N17 and diphenyleneiodonium (a NADPH oxidase inhibitor), indicating that MeJA induces HSP72 expression though HSF1 that is activated via Rac1-NADPH oxidase-independent ROS production pathway. These results suggest that the plant stress hormones share the ability to induce heat shock response in animal cells.

Low glibenclamide concentrations affect endoplasmic reticulum stress in INS-1 cells under glucotoxic or glucolipotoxic conditions.

BACKGROUND/AIMS: ß-Cell apoptosis caused by increased endoplasmic reticulum (ER) stress is an important pathogenic component of type 2 diabetes mellitus. In theory, sulfonylureas, used for the treatment of diabetes, can contribute to ER stress. We assessed changes in ER stress in pancreatic ß-cells under glucotoxic or glucolipotoxic conditions using low concentrations of the sulfonylurea, glibenclamide (GB). METHODS: Low concentrations of GB (10 or 100 nM) were added to INS-1 cells cultured under glucotoxic or glucolipotoxic conditions. The degree of viability, level of apoptosis and levels of markers associated with ER stress were measured. RESULTS: Apoptosis decreased in response to low concentrations of GB under glucolipotoxic but not glucotoxic conditions. Most ER stress markers decreased upon the addition of GB. Under glucotoxic conditions, changes in the levels of ER stress markers were not consistent. However, all decreased significantly under glucolipotoxic conditions. CONCLUSIONS: Low concentrations of GB exerted antiapoptotic effects through the attenuation of ER stress under glucolipotoxic conditions.

The Effects of Glyburide on Apoptosis and Endoplasmic Reticulum Stress in INS-1 Cells in a Glucolipotoxic Condition.

BACKGROUND: ß-cell death due to endoplasmic reticulum (ER) stress has been regarded as an important pathogenic component of type 2 diabetes. The possibility has been suggested that sulfonylurea, currently being used as one of the main oral hypoglycemic agents of type 2 diabetes, increases ER stress, which could lead to sulfonylurea failure. The authors of the present study examined ER stress of ß-cells in a glucolipotoxic condition using glyburide (GB) in an environment mimicking type 2 diabetes. METHODS: Apoptosis was induced by adding various concentrations of GB (0.001 to 200 µM) to a glucolipotoxic condition using 33 mM glucose, and the effects of varied concentrations of palmitate were evaluated via annexin V staining. The markers of ER stress and pro-apoptotic markers were assessed by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction. Additionally, the anti-apoptotic markers were evaluated. RESULTS: Addition of any concentration of GB in 150 µM palmitate and 33 mM glucose did not increase apoptosis. The expression of phosphorylated eukaryotic initiation factor (eIF-2α) was increased and cleaved caspase 3 was decreased by adding GB to a glucolipotoxic condition. However, other ER stress-associated markers such as Bip-1, X-box binding protein-1, ATF-4 and C/EBP-homologous protein transcription factor and anti-apoptotic markers phosphor-p85 phosphatidylinositol 3-kinase and phosphorylation of Akt did not change significantly. CONCLUSION: GB did not show further deleterious effects on the degree of apoptosis or ER stress of INS-1 cells in a glucolipotoxic condition. Increased phosphorylation of eIF-2α may attenuate ER stress for adaptation to increased ER protein load.

The Effect of Glucose Fluctuation on Apoptosis and Function of INS-1 Pancreatic Beta Cells.

BACKGROUND: Blood glucose level continuously fluctuates within a certain range in the human body. In diabetes patients, the extent of such fluctuation is large, despite the strict control of blood glucose. Blood glucose fluctuation has been shown to mediate more adverse effects on vascular endothelial cells and diabetes complications than chronic hyperglycemia, which has been explained as due to oxidative stress. As few previous studies have reported the effects of chronic and intermittent hyperglycemia on the apoptosis and function of pancreatic beta cells, this study reported herein was performed to investigate such effects on these cells. METHODS: For chronic hyperglycemia, INS-1 cells were cultured for 5 days with changes of RPMI 1640 medium containing 33 mM glucose every 12 hours. For intermittent hyperglycemia, the medium containing 11 mM glucose was exchanged with the medium containing 33 mM glucose every 12 hours. Apoptosis was assessed by TUNEL assay Hoechst staining and cleaved caspase 3. Insulin secretory capacity was assessed, and the expression of Mn-SOD and Bcl-2 was measured by Western blotting. RESULTS: In comparison to the control group, INS-1 cells exposed to chronic hyperglycemia and intermittent hyperglycemia showed an increase in apoptosis. The apoptosis of INS-1 cells exposed to intermittent hyperglycemia increased significantly more than the apoptosis of INS-1 cells exposed to chronic hyperglycemia. In comparison to the control group, the insulin secretory capacity in the two hyperglycemic states was decreased, and more with intermittent hyperglycemia than with chronic hyperglycemia. The expression of Mn-SOD and Bcl-2 increased more with chronic hyperglycemia than with intermittent hyperglycemia. CONCLUSION: Intermittent hyperglycemia induced a higher degree of apoptosis and decreased the insulin secretory capacity more in pancreatic beta cells than chronic hyperglycemia. This activity may be mediated by the anti-oxidative enzyme Mn-SOD and the anti-apoptotic signal Bcl-2.