Effects of telmisartan and olmesartan on insulin sensitivity and renal function in spontaneously hypertensive rats fed a high fat diet.

Although telmisartan, an angiotensin II receptor blocker (ARB), has an agonistic action for proliferator-activated receptor (PPAR)-γ in vitro, it remains to be determined whether telmisartan exerts such an action in vivo using a non-toxic dose (<5 mg/kg in rats). To address the issue, telmisartan (2 mg/kg) and olmesartan (2 mg/kg), another ARB without PPAR-γ agonistic action, were given to spontaneously hypertensive rats (SHR) fed a high fat diet (HFD). HFD decreased plasma adiponectin, and caused insulin resistance, hypertriglyceridemia and renal damage, which were improved by ARBs. Protective effects of telmisartan and olmesartan did not significantly differ. In addition, in vitro study showed that 1 µM of telmisartan did not elevate the mRNA expression of adipose protein 2, which is a PPAR-γ-stimulated adipogenic marker gene, in preadipocytes with 3% albumin. To obtain 1 µM of plasma concentration, oral dose of telmisartan was calculated to be 6 mg/kg, which indicates that PPAR-γ agonistic action is negligible with a non-toxic dose of telmisartan (<5 mg/kg) in rats. This study showed that 2 mg/kg of telmisartan and olmesartan ameliorated insulin resistance, hypertriglyceridemia and renal damage in SHR fed a HFD. As beneficial effects of telmisartan and olmesartan did not significantly differ, these were mediated through the PPAR-γ-independent actions.

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.

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.

Effects of atorvastatin and pravastatin on glucose tolerance, adipokine levels and inflammatory markers in hypercholesterolaemic patients.

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. 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. 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 (HbA(1c)) levels. Only obese patients showed increased HbA(1c) levels after atorvastatin treatment. 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.

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, hyperlipidemia, 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 7alpha-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. The first two authors contributed equally to this study.

Rhythmic messenger ribonucleic acid expression of clock genes and adipocytokines in mouse visceral adipose tissue.

Various peripheral tissues show circadian rhythmicity, which is generated at the cellular level by their own core oscillators that are composed of transcriptional/translational feedback loops involving a set of clock genes. Although the circulating levels of some adipocytokines, i.e. bioactive substances secreted by adipocytes, are on a 24-h rhythmic cycle, it remains to be elucidated whether the clock gene system works in adipose tissue. To address this issue, we investigated the daily mRNA expression profiles of the clock genes and adipocytokines in mouse perigonadal adipose tissues. In C57BL/6J mice, all transcript levels of the clock genes (Bmal1, Per1, Per2, Cry1, Cry2, and Dbp) and adipocytokines (adiponectin, resistin, and visfatin) clearly showed 24-h rhythms. On the other hand, the rhythmic expression of these genes was mildly attenuated in obese KK mice and greatly attenuated in more obese, diabetic KK-A(y) mice. Obese diabetes also diminished the rhythmic expression of the clock genes in the liver. Interestingly, a 2-wk treatment of KK and KK-A(y) mice with pioglitazone impaired the 24-h rhythmicity of the mRNA expression of the clock genes and adipocytokines despite the antidiabetic effect of the drug. In contrast, pioglitazone improved the attenuated rhythmicity in the liver. These findings suggest that the intracellular clock gene system acts in visceral adipose tissues as well as liver and is influenced by the conditions of obesity/type 2 diabetes and pioglitazone treatment.

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.

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. METHODS: Nifedipine oxidation, activity and protein expression of NADPH-cytochrome reductase (POR) of HepG2-GS-3A4 cell were measured. CO-binding spectrumassay in microsomal fraction of the cells was also evaluated. KEY FINDINGS: DHB and ketoconazole, a well-known inhibitor of CYP3A4, inhibited nifedipine oxidation in a concentration-dependent manner. DHB at a concentration of 3.0 µm, sufficient to inhibit the nifedipine oxidation, decreased POR activity; however, ketoconazole at a concentration of 0.9 µm, sufficient to inhibit the oxidation, did not affect the activity. The expression of POR protein in HepG2-GS-3A4 cells was not changed by either DHB or ketoconazole. The expression of CYP3A4 mRNA and protein was not changed by the addition of DHB or ketoconazole. DHB also reduced the absorption rate at 450 nm in a CO-binding spectrum assay without alteration of the wavelength of maximum absorption. The mean absorption value at 450 nm slightly decreased with ketoconazole; however, the difference was not significant. CONCLUSIONS: We concluded that inhibition of CYP3A4 activity by DHB includes the inhibition of POR activity. HepG2-GS-3A4 might be a good tool to evaluate the mechanisms.

Profile of rhythmic gene expression in the livers of obese diabetic KK-A(y) mice.

Although a number of genes expressed in most tissues, including the liver, exhibit circadian regulation, gene expression profiles are usually examined only at one scheduled time each day. In this study, we investigated the effects of obese diabetes on the hepatic mRNA levels of various genes at 6-h intervals over a single 24-h period. Microarray analysis revealed that many genes are expressed rhythmically, not only in control KK mice but also in obese diabetic KK-A(y) mice. Real-time quantitative PCR verified that 19 of 23 putative circadianly expressed genes showed significant 24-h rhythmicity in both strains. However, obese diabetes attenuated these expression rhythms in 10 of 19 genes. More importantly, the effects of obese diabetes were observed throughout the day in only two genes. These results suggest that observation time influences the results of gene expression analyses of genes expressed circadianly.

Effects of grapefruit juice on the pharmacokinetics of pitavastatin and atorvastatin.

AIMS: To compare the effects of grapefruit juice (GFJ) on the pharmacokinetics of pitavastatin and atorvastatin. METHODS: In a randomized, four-phase crossover study, eight healthy subjects consumed either GFJ or water t.i.d. for 4 days in each trial. On each final day, a single dose of 4 mg pitavastatin or 20 mg atorvastatin was administered. RESULTS: GFJ increased the mean AUC(0-24) of atorvastatin acid by 83% (95% CI 23-144%) and that of pitavastatin acid by 13% (-3 to 29%). CONCLUSIONS: Pitavastatin, unlike atorvastatin, appears to be scarcely affected by the CYP3A4-mediated metabolism.

Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase.

Depolarization of the sarcolemma of smooth muscle cells activates voltage-gated Ca2+ channels, influx of Ca2+ and activation of cross-bridge cycling by phosphorylation of myosin catalysed by Ca2+/calmodulin-dependent myosin light-chain kinase (MLCK). Agonist stimulation of smooth muscle contraction often involves other kinases in addition to MLCK. In the present study, we address the hypothesis that membrane depolarization-induced contraction of rat caudal arterial smooth muscle may involve activation of Rho-associated kinase (ROK). Addition of 60 mM K+ to de-endothelialized muscle strips in the presence of prazosin and propranolol induced a contraction that peaked rapidly and then declined to a steady level of force corresponding to approx. 30% of the peak contraction. This contractile response was abolished by the Ca2+-channel blocker nicardipine or the removal of extracellular Ca2+. An MLCK inhibitor (ML-9) inhibited both the phasic and tonic components of K+-induced contraction. On the other hand, the ROK inhibitors Y-27632 and HA-1077 abolished the tonic component of K+-induced contraction, and slightly reduced the phasic component. Phosphorylation levels of the 20-kDa light chain of myosin increased rapidly in response to 60 mM K+ and subsequently declined to a steady-state level significantly greater than the resting level. Y-27632 abolished the sustained and reduced the phasic elevation of the phosphorylation of the 20-kDa light chain of myosin, without affecting the K+-induced elevation of cytosolic free Ca2+ concentration. These results indicate that ROK activation plays an important role in the sustained phase of K+-induced contraction of rat caudal arterial smooth muscle, but has little involvement in the phasic component of K+-induced contraction. Furthermore, these results are consistent with inhibition of myosin light-chain phosphatase by ROK, which would account for the sustained elevation of myosin phosphorylation and tension in response to membrane depolarization.