Hayato Yanagihara

High‐Fat Feeding Exerts Minimal Effects on Rhythmic mRNA Expression of Clock Genes in Mouse Peripheral Tissues

Recent studies have suggested that the impairment of the circadian molecular clock in peripheral tissues, including adipose tissue, is involved in the development of metabolic syndrome. Although the disorder is often caused by dietary obesity, it remains to be elucidated whether dietary obesity or high‐caloric intake per se affects the molecular clock system. To address this issue, this study investigated the effect of high‐fat feeding on the rhythmic mRNA expression of clock genes (Clock, Bmal1, Per1, Per2, Cry1, Cry2, and Dbp) in mouse visceral adipose tissue and liver. Mice fed a high‐fat diet for 8 wks developed a mild but overt metabolic syndrome of obesity, hyperlipidemina, and hyperglycemia. However, the high‐fat feeding had only minimal effects on the rhythmic expression of the clock genes examined in both tissues. On the other hand, daily rhythmicity in the transcript level of cholesterol 7α‐hydroxylase, a hepatic enzyme controlling circadian cholesterol homeostasis, disappeared in the mice on high‐fat chow. These results suggest that high‐fat feeding and mild metabolic syndrome scarcely alter the molecular clock system in mouse peripheral tissues, and that physiological circadian rhythms could be affected without altering the system. Further studies are needed to better understand the role of the circadian molecular clock in the development of metabolic syndrome.

Daily Rhythms of P‐glycoprotein Expression in Mice

Recent studies have shown the gene expression of several transporters to be circadian rhythmic. However, it remains to be elucidated whether the expression of P‐glycoprotein, which is involved in the transport of many medications, undergoes 24 h rhythmicity. To address this issue, we investigated daily profiles of P‐glycoprotein mRNA and protein levels in peripheral mouse tissues. In the liver and intestine, but not in the kidney, Abcb1a mRNA expression showed clear 24 h rhythmicity. On the other hand, Abcb1b and Abcb4, the other P‐glycoprotein genes, did not exhibit significant rhythmic expression in the studied tissues. In the intestine, levels of whole P‐glycoprotein also exhibited a daily rhythm, with a peak occurring in the latter half of the light phase and a trough at the onset of the light phase. Consistent with the day‐night change of P‐glycoprotein level, the ex vivo accumulation of digoxin, an Abcb1a P‐glycoprotein substrate, into the intestinal segments at the onset of dark phase was significantly lower than it was at the onset of the light phase. Thus, Abcb1a P‐glycoprotein expression, and apparently its function, are 24 h rhythmic at least in mouse intestine tissue. This circadian variation might be involved in various chronopharmacological phenomena.

EFFECTS OF ATORVASTATIN AND PRAVASTATIN ON GLUCOSE TOLERANCE, ADIPOKINE LEVELS AND INFLAMMATORY MARKERS IN HYPERCHOLESTEROLAEMIC PATIENTS

1 Several randomized clinical trials have suggested that atorvastatin and pravastatin may differ in terms of their pleiotropic effects. To verify this, we compared the effects of both statins on glucose tolerance, adipokine concentrations and inflammatory markers. 2 A total of 36 hypercholesterolaemic patients without known coronary heart disease (CHD) were enrolled in an open‐label, randomized, crossover study. The patients received pravastatin or atorvastatin (10 mg/day) for 4 months and then switched to the other statin for an additional 4 months. 3 At the end of both treatment periods, atorvastatin significantly reduced the concentration of serum lipids (total and low‐density lipoprotein–cholesterol and triglycerides) and inflammatory markers (high‐sensitivity C‐reactive protein and tumour necrosis factor‐a) and increased serum adiponectin levels compared with pravastatin treatment. Although these effects would be expected to improve insulin sensitivity, atorvastatin did not affect glucose tolerance, which was assessed by fasting glucose and insulin concentrations, the homeostasis model assessment index and glycosylated haemoglobin (HbA1c) levels. Only obese patients showed increased HbA1c levels after atorvastatin treatment. 4 Our results suggest that atorvastatin has both advantages and disadvantages compared with pravastatin treatment. Further studies are required to compare the relative clinical value of atorvastatin and pravastatin, especially in obese patients without CHD.

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.

Influence of a Time-Restricted Feeding Schedule on the Daily Rhythm of abcb1a Gene Expression and Its Function in Rat Intestine

P-glycoprotein (P-gp) is one of the ATP-binding cassette transporters and acts as an efflux pump for cytotoxic substances. P-gp mRNA expression and transporting activity show the daily rhythm and contribute to the chrono-pharmacokinetic profiles of many drugs. It is reported that the daily rhythm of abcb1a mRNA is regulated by a circadian clock-controlled output pathway. Time-restricted feeding is well known to shift the peripheral circadian phase of clock gene expression without changing the central clock function. This study was undertaken to examine the influence of a time-restricted feeding procedure during the light phase on the daily rhythms of abcb1a mRNA expression and P-gp activity. The abcb1a mRNA and P-gp activity showed a daily rhythm with a peak early in the dark phase in rat intestine under ad libitum feeding. Time-restricted feeding during the light phase shifted these rhythms to 12-h advance. The mRNA expression of clock genes (DBP and HLF, the transcript activators of abcb1a) also showed daily rhythms, and their phases were shifted by the time-restricted feeding procedure. The peak time of DBP mRNA expression was similar to that of abcb1a mRNA expression under ad libitum feeding and time-restricted feeding conditions. These results indicate that a time-restricted feeding procedure changes DBP mRNA expression, which in turn influences abcb1a mRNA expression and P-gp activity.

Human CYP3A4-introduced HepG2 cells: In vitro screening system of new chemicals for the evaluation of CYP3A4-inhibiting activity

1. The aims were to attest whether HepG2-GS-3A4, a cell line into which the human CYP3A4 gene was introduced, can be used for a screening of chemicals that will inhibit CYP3A4 activity. 2. The capacity of the cells for metabolizing CYP3A4 substrates in vitro was evaluated. Also determined was the effect of CYP3A4 inhibitors and non-inhibitors on nifedipine hydroxylation. Western blot, immunohistochemostry and determination of β-nicotinamide adenine dinucleotide phosphate (NADPH)-reductase activity were performed. 3. HepG2-GS-3A4 selectively metabolized substrates of CYP3A4 (diazepam, nordiazepam, lidocaine, atorvastatin, and nifedipine) to a greater degree than control. The metabolites were easily detected in the culture medium. Values of Vmax of HepG2-GS-3A4 were about 30- to 100-fold higher than those of the control, while values of Km were comparable. Pre-incubation of cimetidine and ketoconazole significantly inhibited nifedipine hydroxylation, while addition of inhibitors specific to other isoforms of CYPs had no substantial effect. The HepG2-GS-3A4 expressed a higher amount of CYP3A4 protein and mRNA than control. Most NADPH reductase activity was detected in microsomal fractions. 4 In conclusion, HepG2-GS-3A4 sufficiently and selectively metabolize substrates of CYP3A4, and inhibitors of CYP3A4 reduced the metabolism. Because the metabolites were easily detected in the culture medium, this cell might be useful for the new and easy screening of new drugs for the evaluation of CYP3A4-inhibiting activity in vitro.

Inhibition of CYP3A4 by 6',7'-dihydroxybergamottin in human CYP3A4 over-expressed hepG2 cells.

Objectives  We previously established HepG2‐GS‐3A4, a cell line from hepatoblastoma with overexpression of human CYP3A4 and glutamine synthetase (GS). We further reported that these cells can be applied for screening inhibitors of CYP3A4 in vitro. The purpose of this study was to determine whether our CYP3A4‐overexpresed cell could be applied to evaluate mechanisms of CYP3A4 inhibition by 6′,7′‐dihydroxybergamottin (DHB), which is one of the major furanocoumarins in grapefruit juice, by using these cells.