Kenji Iizuka

Can Exercise Training With Weight Loss Lower Serum C-Reactive Protein Levels?

Objective—C-reactive protein (CRP), an obesity-related inflammatory marker, is a promising predictor for cardiovascular disease and may be a mediator for atherogenesis. It has been reported that diet-induced weight loss lowered CRP levels. However, the effect of exercise training, another therapy that can reduce weight, on CRP is still unclear. We examined effects of exercise training with weight loss on CRP levels and conventional cardiovascular risks. Methods and Results—A total of 227 apparently healthy women were recruited, and 199 subjects (average age 52 years) completed a 2-month weight reduction program consisting of supervised aerobic exercises. After the program, weight was reduced from 65.8 to 62.8 kg (P<0.0001), and all conventional variables were remarkably improved. Similarly, CRP levels were significantly decreased, from 0.63 (0.28 to 1.19) to 0.41 (0.18 to 0.80) mg/L (P<0.0001). However, in contrast to other variables, the changes in CRP levels were not proportionally associated with the extent of weight reduction. In the quartile analysis of % weight reduction, the largest weight reduction quartile did not show significant decreases in CRP levels, whereas moderate quartile showed remarkable CRP decreases. Conclusions—Exercise training with weight reduction disproportionately lowered CRP levels. Considering inflammatory status, there might be an optimal pace of exercise with weight loss.

Phosphatidylinositol metabolism in hypertrophic rat heart.

The accumulation of inositol 1,4,5-trisphosphate (IP3) after hormonal stimulation has a physiological role, possibly by alteration of Ca2+ levels in cardiac myocyte. However, this accumulation has not been studied under pathophysiological conditions. In this report, we examine phosphatidylinositol metabolism during cellular response to norepinephrine in pressure-overloaded hypertrophic rat heart. After stimulation with norepinephrine, the accumulations of IP3 and diacylglyceride significantly increased in isolated myocytes from stroke-prone spontaneously hypertensive rat (SHRSP) heart, indicating phosphatidylinositol-specific phospholipase C activity increased in SHRSP heart cells. Protein kinase C activity was also enhanced in SHRSP, with a marked increase in particulate activity. We determined the intracellular calcium concentration and found it to be higher in SHRSP than in Wistar-Kyoto (WKY) rats at 30-40 weeks of age. Ca2+ influx was also elevated in SHRSP stimulated by norepinephrine. In SHRSP heart, cytosolic Ca2+ concentration may rise quickly in response to some stimuli, such as alpha 1-adrenergic stimulation, which is shown to be one of the pathways that increases cytosolic Ca2+ levels in hypertrophied rat heart. These data suggest that a part of the phosphatidylinositol-turnover pathway, such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway, may play an important role in the development of hypertrophy in SHRSP heart.

Calpain is activated during hypoxic myocardial cell injury

Cell death during hypoxia rose to 80% after 6 h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6 h. This activity was markedly inhibited by calpain-specific inhibitor I (n-acetyl-leucyle-leucyle-norleucinal). beta-Adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. On the other hand, alpha-adrenergic blocking agents did not affect calpain activity and cell death under hypoxic conditions. These results prove that beta-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.

Phospholipid metabolism and prostacyclin synthesis in hypoxic myocytes

We observed that in hypoxic myocardial cells prostacyclin and arachidonic acid release increased and that during hypoxia phospholipid degradation also occurred. In order to clarify the mechanism of phospholipid degradation, we determined the activity of phospholipases A2 and C. We found that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were markedly decreased and that lysophosphatidylcholine and lysophosphatidylethanolamine were increased. In contrast, there was only slight phosphatidylinositol degradation and no lysophosphatidylinositol elevation was observed. These results show that phospholipase A2 was activated in hypoxic myocytes and had substrate specificity towards PC and PE. To study phospholipase C activity, membrane phospholipids were labeled with [3H]choline, [3H]inositol or [3H]ethanolamine. The release of inositol was observed, but neither choline nor ethanolamine was released. In hypoxia, myocardial-cell phospholipase C has high substrate specificity towards phosphatidylinositol. The activation of phospholipases is closely related to the intracellular Ca2+ concentration; it is though that inositol polyphosphatides may regulate intracellular Ca2+. We determined how Ca2+ influx occurs in hypoxia. beta-Adrenergic blockade and Ca2+ antagonists markedly suppressed Ca2+ influx, phospholipase A2 activity, phospholipase C activity and cell death. However, the alpha 1-adrenergic blockade was less effective in suppressing these phenomena. These results suggest that in hypoxic myocardial cells Ca2+ influx mediated by beta-adrenergic stimulation activates phospholipases A2 and C, and that phospholipid degradation and prostacyclin release then occur.

Increased Calcium Release from Sarcoplasmic Reticulum Stimulated by Inositol Trisphosphate in Spontaneously Hypertensive Rat Heart Cells

It is known that inositol (1, 4, 5)-trisphosphate (IP3) stimulates Ca2+ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP3 on Ca2+ release from SR in cardiac myocytes. The effect of IP3 on Ca2+ release from SR in hypertrophic cardiac cells was also determined.We examined the effects of IP3 on Ca2+ release from cardiac myocyte SR by the bigital-image method in a single cell. We also determined the effect of IP3 on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with45Ca2+, and then incubated with the indicated concentrations of IP3 for 1 min at 37°C. In cardiac myocytes treated with saponin, Ca2+ release stimulated by 10 μM (1, 4, 5)-IP3 was detected by fura-2. In45Ca2+ prelabeled SR, the maximal Ca2+ release was achieved at 10 μM IP3 incubated for 1 min. The release of Ca2+ was higher in Sr of SHR than in the SR of WKY. IP3 stimulates Ca2+ release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.

N-terminal kinase, and c-Src are activated in human aortic smooth muscle cells by pressure stress

Mechanical forces related to pressure and flow are important for cell hypertrophy and proliferation. There are still a few studies that examine responses of human vascular smooth muscle cells to pure pressure stress (transmural pressure). It is unclear as to which mechanisms are involved in cellular responses to pressure elevation. On the other hand, although the involvement of the local renin-angiotensin system (RAS) in pressure-induced responses was reported, the results were contradictory. It still remains to be determined whether RAS in human vascular smooth muscle cells is activated by pure pressure stress. We studied the upstream signal transduction events of extracellular signal kinase (ERK) in response to atmospheric pressure stress and involvement of angiotensin II in pressure-induced cell proliferation in human aortic smooth muscle cells (HASMC). A pressure-loading apparatus was set up to examine the effects of atmospheric pressure on human aortic smooth muscle cells. Pressure application of 160 mmHg for 3 h produced cell proliferation and activated ERK and c-JUN N-terminal kinase (JNK). ACE inhibitor suppressed all of them. ERK kinase (MEK) inhibitor also suppressed cell proliferation stimulated by pure pressure. The phosphorylated c-Src was increased by pure pressure stress. The treatment with c-Src kinase inhibitor suppressed pressure-induced proliferative response. In summary, our study found that ERK activation mediated pure pressure-induced proliferative response of HASMC. This activation was partly mediated by c-Src. (Mol Cell Biochem 262: 71–78, 2004)

Translocation of G-Protein β3 subunit from the cytosol pool to the membrane pool by β1-Adrenergic receptor stimulation in perfused rat hearts

To elucidate the intracellular function and localization of the heterotrimeric G-protein beta3 subunit (Gbeta3) in the heart, we studied the effects of subtype-specific beta-adrenergic receptor (beta-AR) stimulation on Gbeta3 localization using isoform-specific antibodies. The amount of Gbeta3 in the cytosol dramatically decreased in hearts perfused with isoproterenol (ISO) alone or ISO with ICI 118551, a beta2-AR antagonist. Propranolol or CGP 20712A, a beta1-AR antagonist, blocked the ISO-induced decrease in the Gbeta3 content of the cytosol. In contrast, Gbeta3 content of the membrane fraction significantly increased in hearts perfused with ISO alone or ISO with ICI 118551. We conclude that stimulation of the beta1-AR induces isoform-specific translocation of Gbeta3 from the cytosol to the membrane fraction in rat hearts.

Inositol trisphosphate kinase activity in hypertrophied rat heart.

In the present experiment, we demonstrated that IP3 kinase activity was increased in SHRSP heart compared to WKY heart. IP3 kinase activity in the heart was highest in the cytosolic fraction in both SHRSP and WKY. Its activity progressively increased with age in 5- to 20-week SHRSP. The activity reached about three times the level of 5-week-old SHRSP in 40-week-old SHRSP. On the other hand, in WKY it was 1.3-fold at 40 weeks compared with that at 5 weeks. We determined the effect of divalent cations on IP3 kinase activity. Ca2+ stimulated its activity in a dose-dependent manner at 10(-9) to 10(-6) M. In SHRSP it was enhanced about 2.1-fold at 10(-6) M of Ca2+, but in WKY it was 1.5-fold at 1.0(-6) M of Ca2+. Mn2+ also stimulated IP3 kinase activity in both groups of animals, while, Fe2+, Zn2+, and Cu2+ inhibited IP3 kinase activity. In our experiment IP3 kinase activity was increased in SHRSP and its activity was markedly affected by divalent cations. These data suggest that the accumulations of IP3 and IP4 after hormonal stimulation play a physiologic role, possibly by alteration of Ca2+ levels in cardiac tissue.

Pure pressure stress increased monocarboxylate transporter in human aortic smooth muscle cell membrane

Lactate is formed and utilized continuously under fully aerobic conditions. Lactate is oxidized actively at all times, especially during exercise. Family of monocarboxylate transport proteins (MCTs) that are differentially expressed in cells and tissues accomplishes the facilitated transport of lactate across membranes. Previously we reported that there is MCT1 in blood circulation. We also reported the pressure stress stimulated cell proliferation in aortic smooth muscle cells (HASMC). In this experiment we attempted to prove the existence of MCT1 in HASMC and to clarify the effect of pressure stress on MCT1 localization in HASMC. We determined succinate dehydrogenase (SDH) activity as a marker of energy metabolism in cells. SDH activity was increased by pressure stress. Lactate enhanced the SDH activity under pressure stress (160 mmHg for 3 h) as dose dependent manner. On the other hand, lactate excretion was suppressed by the addition of lactate. We could detect MCT1 in the cytosolic and the membrane fractions of HASMC. The pressure stress increased MCT1 in the membrane fraction in the presence of extracellular lactate. In summary, we proved the existence of MCT1 in HASMC. Pressure stress changed the localization of MCT1. The increased membranous MCT1 may transport lactate for energy metabolism in cells.

The leakage of fatty acid binding protein from cultured myocardial cells during hypoxia

SummaryFatty-acid binding protein (FABP) is thought to play an important role as a carrier protein of fatty acids in cells. It may leak from damaged cells, because its molecular weight is low (mol wt 14000) and it accounts for several percent of soluble protein. In this experiment we attempted to use FABP as a marker of cell injury under hypoxia in cultured myocytes. Newborn-rat myocytes were incubated under hypoxic treatment for 6 hours, and then the releases of FABP and CPK were measured. The cell-death ratio during hypoxygenation increased from 4 hours and rose to 80% at 6 hours, but it was only 8% under aerobic conditions. FABP in medium was detected at 1 hour, and rapidly increased and reached a plateau at 4 hours. On the other hand, CPK in medium was negligible during the 3 hours, then slightly increased. Ca antagonists and a β1 blocking agent inhibited the release of FABP and prevented cell death. But the α1-adrenergic blocking agent had little effect on preventing FABP leakage and cell death. These results show that FABP is of use as a marker of myocardial cell injury and revealed that the Ca antagonist and β1 blocking agent are useful drugs for the protection of myocardial cell injury in hypoxia.