Kentarou Ushijima

Impairment of Peripheral Circadian Clocks Precedes Metabolic Abnormalities in ob/ob Mice

Recent studies have demonstrated relationships between the dysfunction of circadian clocks and the development of metabolic abnormalities, but the chicken-and-egg question remains unresolved. To address this issue, we investigated the cause-effect relationship in obese, diabetic ob/ob mice. Compared with control C57BL/6J mice, the daily mRNA expression profiles of the clock and clock-controlled genes Clock, Bmal1, Cry1, Per1, Per2, and Dbp were substantially dampened in the liver and adipose tissue, but not the hypothalamic suprachiasmatic nucleus, of 10-wk-old ob/ob mice. Four-week feeding of a low-calorie diet and administration of leptin over a 7-d period attenuated, to a significant and comparable extent, the observed metabolic abnormalities (obesity, hyperglycemia, hyperinsulinemia, and hypercholesterolemia) in the ob/ob mice. However, only leptin treatment improved the impaired peripheral clocks. In addition, clock function, assessed by measuring levels of Per1, Per2, and Dbp mRNA at around peak times, was also reduced in the peripheral tissues of 3-wk-old ob/ob mice without any overt metabolic abnormalities. Collectively these results indicate that the impairment of peripheral clocks in ob/ob mice does not result from metabolic abnormalities but may instead be at least partially caused by leptin deficiency itself. Further studies are needed to clarify how leptin deficiency affects peripheral clocks.

Chronobiological disturbances with hyperthermia and hypercortisolism induced by chronic mild stress in rats.

The chronic mild stress (CMS) model has been established as a realistic model of depressive disorder as it simulates anhedonia. In the present study, the consumption of sucrose solution was decreased in the rats exposed to CMS, which coincided with many published studies. Since depression is a multifaceted disorder, and a number of symptoms may be present, including circadian rhythm disturbances, we attempted to find the chronobiological abnormalities in CMS rats. After 4-week of the stress procedure, the rhythmic pattern of rectal temperature in the CMS group was extinguished. In particular, the temperature in the CMS group in the light phase was significantly higher than that in the control group. The plasma corticosterone levels in the CMS group were remarkably increased in the light phase compared to the control group, but not in the dark phase. It was concluded that the CMS procedure caused the disturbance of circadian rhythms with hyperthermia and hypercortisolism.

Differential impacts of CYP2C19 gene polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine.

We examined the influence of CYP2C19 polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine. The platelet aggregation induced by 20 µmol/l adenosine diphosphate (ADP) and CYP2C19 single‐nucleotide polymorphisms (*2 and *3) was determined in patients with coronary artery disease (CAD) who were taking aspirin alone (n = 21), aspirin plus clopidogrel (n = 97), or aspirin plus ticlopidine (n = 47). The degree of platelet aggregation in the clopidogrel group, although not in the ticlopidine group, depended on the CYP2C19 polymorphism, and the maximal platelet aggregation in poor metabolizers (PMs) taking clopidogrel was equivalent to that in the group taking aspirin alone. After being switched from clopidogrel to ticlopidine, all seven of the PMs showed markedly lower platelet aggregation. These results suggest that CYP2C19 polymorphisms have a profound impact on the antiplatelet effect of clopidogrel but not on that of ticlopidine. Ticlopidine may be an effective therapeutic option for CYP2C19 PMs.

Protective effect of amlodipine against osteoporosis in stroke-prone spontaneously hypertensive rats.

Hypertensive patients have an increasing risk of osteoporosis. A recent case-controlled study has demonstrated that anti-hypertensive therapy reduced a risk of fracture in these patients. In this study, we investigated whether amlodipine protects against the reduction in bone density in stroke-prone spontaneously hypertensive rats (SHR-sp). Oral dosing of amlodipine (0.5 and 3.0mg/kg/day) was started when SHR-sp were 3 months old, and continued for 3 months. At the end of the experiment, bone density of femur and serum concentrations of calcium, parathyroid hormone (PTH) and C-telopeptide of type I collagen (CTx), reflecting osteoclast activity, were measured. The bone density dose-dependently increased by the treatment with amlodipine. In addition, amlodipine reduced serum concentrations of calcium, PTH and CTx. This study showed that amlodipine prevents the reduction in bone density during the repeated dosing in SHR-sp. Amlodipine might exert its effect through a direct inhibition of osteoclast function and/or suppression of PTH secretion and subsequent inhibition of osteoclast activity.

ASSOCIATIONS OF METABOLIC PARAMETERS AND ETHANOL CONSUMPTION WITH MESSENGER RNA EXPRESSION OF CLOCK GENES IN HEALTHY MEN

Recent studies suggest that the impairment of circadian clock function causes various pathological conditions, such as obesity, diabetes, and alcoholism, and an altered mRNA expression of clock genes was found under these conditions. However, it remains to be determined whether clock gene expression varies depending on metabolic conditions even in healthy people. To address this issue, we investigated the associations of metabolic parameters and alcohol consumption with mRNA expression of clock genes (CLOCK, BMAL1, PER1, PER2, and PER3) in peripheral blood cells obtained from 29 healthy non-obese elderly men (age 51–78 yrs) who adhered to a regular sleep-wake routine, through a single time-of-day venous blood sampling at ∼09:00 h. There were significant correlations between (1) waist circumference and mRNA level of PER1 (r =−0.43), (2) plasma glucose concentration and PER2 (r =−0.50), (3) ethanol consumption and BMAL1 (r =−0.43), and (4) serum γ-GTP concentration (a sensitive marker of alcohol consumption) and PER2 (r =−0.40). These results suggest mRNA expression of clock genes is associated with obesity, glucose tolerance, and ethanol consumption even in healthy people. (Author correspondence: akiofuji@jichi.ac.jp)

Clock Gene Expression in the Liver and Adipose Tissues of Non-Obese Type 2 Diabetic Goto-Kakizaki Rats

Recent studies have revealed a close relationship between the pathophysiology of metabolic syndrome, which is characterized by obesity and hyperglycemia, and the functioning of internal molecular clocks. In this study, we show that the rhythmic mRNA expression of clock genes (Clock, Bmal1, Cry1, and Dbp) is not attenuated in the liver and visceral adipose tissues of Goto-Kakizaki rats, a model of nonobese, type 2 diabetes, as compared to control Wistar rats. Our results suggest that molecular clock impairment in peripheral tissues of obese diabetic animals may be either caused by obesity-related factor(s), but not hyperglycemia, or be a cause, but not a consequence, of hyperglycemia.

Cranberry juice suppressed the diclofenac metabolism by human liver microsomes, but not in healthy human subjects

AIM To investigate a potential interaction between cranberry juice and diclofenac, a substrate of CYP2C9. METHODS The inhibitory effect of cranberry juice on diclofenac metabolism was determined using human liver microsome assay. Subsequently, we performed a clinical trial in healthy human subjects to determine whether the repeated consumption of cranberry juice changed the diclofenac pharmacokinetics. RESULTS Cranberry juice significantly suppressed diclofenac metabolism by human liver microsomes. On the other hand, repeated consumption of cranberry juice did not influence the diclofenac pharmacokinetics in human subjects. CONCLUSIONS Cranberry juice inhibited diclofenac metabolism by human liver microsomes, but not in human subjects. Based on the present and previous findings, we think that although cranberry juice inhibits CYP2C9 activity in vitro, it does not change the pharmacokinetics of medications metabolized by CYP2C9 in clinical situations.

Tissue-Dependent Alterations of the Clock Gene Expression Rhythms in Leptin-Resistant Zucker Diabetic Fatty Rats

Recent studies have demonstrated that circadian clocks are impaired in liver and adipose tissue of both leptin-deficient ob/ob and leptin-resistant KK-Ay mice. Because impairment of peripheral clocks precedes metabolic abnormalities in ob/ob mice, leptin signaling might be important for modulating peripheral clocks. To assess this hypothesis, the authors determined daily mRNA expression profiles of clock genes Clock, Arntl, Per1, Per2, Cry1, Dbp, and Nr1d1 in several tissues of leptin-receptor-deficient Zucker diabetic fatty (ZDF) rats. Transcript levels of some of these genes around the respective peak times decreased significantly in the liver, but not in the suprachiasmatic nucleus, mesenteric adipose tissue, and heart, compared to those in control rats. In contrast, mRNA levels of Per1 and Dbp around the peak time increased in the aorta of ZDF rats. However, expression rhythms of these clock genes in serum-stimulated cultured cells isolated from the aorta of ZDF rats were quite similar to those in serum-stimulated aortic cells of control rats. These results show that systemic leptin signaling defect influences peripheral clocks in a tissue-dependent manner, suggesting the possibility that leptin indirectly modulates the clocks in at least a subset of peripheral tissues. (Author correspondence: akiofuji@jichi.ac.jp)

Influence of Age on Clock Gene Expression in Peripheral Blood Cells of Healthy Women

Recent studies have demonstrated a close relationship between circadian clock function and the development of obesity and various age-related diseases. In this study, we investigated whether messenger RNA (mRNA) levels of clock genes are associated with age, body mass index, blood pressures, fasting plasma glucose, or shift work. Peripheral blood cells were obtained from 70 healthy women, including 25 shift workers, at approximately 9:00 AM. Transcript levels of clock genes (CLOCK, BMAL1, PER1, and PER3) were determined by real-time quantitative polymerase chain reaction. Stepwise multiple regression analysis demonstrated that BMAL1 mRNA levels were correlated only with age (beta = -.50, p < .001). In contrast, PER3 levels were correlated with fasting plasma glucose (beta = -.29, p < .05) and shift work (beta = .31, p < .05). These results suggest that increased age, glucose intolerance, and irregular hours independently affect the intracellular clock in humans.

Drug interaction between celecoxib and methotrexate in organic anion transporter 3–transfected renal cells and in rats in vivo

Methotrexate has a clinically important pharmacokinetic interaction with nonsteroidal anti-inflammatory drugs (NSAIDs) mainly through its competition for tubular secretion via the renal organic anion transporter 3 (OAT3). We have previously reported the usefulness of OAT3-transfected renal tubular cells for screening of the drugs which interfere with the pharmacokinetics of methotrexate. Celecoxib, a cyclooxygenase (COX) 2 inhibitor, has not been reported to interact with methotrexate, but the mechanisms are unclear why the interaction did not occur. The purpose of this study was to evaluate the effect of celecoxib on methotrexate tubular secretion using a renal cell line stably expressing human OAT3 (S2-hOAT3), and to evaluate the pharmacokinetic interaction of the two drugs in rats. [3H]methotrexate uptake into S2-hOAT3 cells was significantly inhibited by celecoxib in a concentration-dependent manner and the Ki value was 35.3 microM. However, methotrexate serum concentrations and urinary excretion of methotrexate over 24 h in rats were not affected by celecoxib (50, 200 mg/kg). Celecoxib serum concentrations were increased by the increase in celecoxib dosage and the maximum drug concentration (Cmax) was 20.6 microM (celecoxib 200 mg/kg), which did not reach the Ki value obtained in the in vitro study. These results indicated that celecoxib inhibited the secretion of methotrexate via hOAT3, which suggested that celecoxib was a substrate of hOAT3. However, co-administration of the two drugs at clinical dosage did not affect the pharmacokinetics of methotrexate, because the serum concentrations did not reach the Ki value. Although the accumulation study using S2-hOAT3 cells was useful to predict the interaction between the new drug and methotrexate in vivo, a comparison of the Ki value with the Cmax in clinical dosage was necessary to evaluate the degree of this interaction.