Hitoshi Ando

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.

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.

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model. Methodology/Principal Findings Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH. Conclusions/Significance These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.

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.

Urinary vanin-1 as a novel biomarker for early detection of drug-induced acute kidney injury

Drug-induced nephrotoxicity is a serious problem in patients with hospital-acquired acute kidney injury (AKI). A new renal biomarker is needed because traditional markers are not sensitive for early detection of drug-induced AKI. In a recent study, we demonstrated that vanin-1 is a novel candidate biomarker of nephrotoxicant-induced kidney injury. The objective of the present study is to determine whether the increase in urinary vanin-1 is detected before the elevations of serum creatinine or urinary N-acetyl-β-glucosaminidase (NAG), kidney injury molecule-1 (Kim-1), and neutrophil gelatinase-associated lipocalin (NGAL) in the two well established animal models of drug-induced AKI. After the administration of a higher dose of cisplatin (10 mg/kg, a single intraperitoneal dose) or gentamicin (120 mg/kg per day, once daily intraperitoneal dose for 9 days), urinary vanin-1 was detected earlier than the other biomarkers. In rats treated with a lower dose of cisplatin (5 mg/kg, a single intraperitoneal dose) or gentamicin (40 mg/kg per day, once daily intraperitoneal dose for 9 days), serum creatinine and urinary NAG were not changed throughout the study period, whereas urinary vanin-1, Kim-1, and NGAL were significantly increased. The renal vanin-1 protein levels were significantly decreased in rats treated with the higher dose of cisplatin on day 5 and gentamicin on day 9, and the immunofluorescence analyses confirmed that vanin-1 immunoreactivity in tubular cells was reduced with the time after the dose of cisplatin, indicating that urinary vanin-1 was leaked from tubular cells. These results suggest that, compared with urinary Kim-1 and NGAL, urinary vanin-1 is an earlier and equally sensitive biomarker for drug-induced AKI.

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.

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.

Vanin-1; a potential biomarker for nephrotoxicant-induced renal injury

Because traditional markers for detecting renal injury are generally insensitive and nonspecific, we tried to identify some useful biomarkers. Microarray analyses and quantitative real-time PCR using human renal tubular cells showed that the mRNA expression of VNN-1 which encodes vanin-1, increased after the exposure of these cells to organic solvents (allyl alcohol, ethylene glycol, formaldehyde, chloroform, and phenol) for 24h. The mRNA levels of other inflammation-related molecules such as monocyte chemoattractant protein 1 (MCP-1) and kidney injury molecule-1 (KIM-1) also increased after the exposure to organic solvents, although their elevations were slower than that of vanin-1. In rats treated with ethylene glycol for 3 weeks, tubular injury was detected by histological examination, but not by traditional biomarkers including serum creatinine and urinary N-acetyl-β-glucosaminidase. The mRNA levels of vanin-1 and Kim-1, but not MCP-1, significantly elevated in the renal cortices of ethylene glycol-exposed rats. On immunofluorescence analyses, vanin-1 signal was detected specifically in the renal tubules with a remarkable expression in the ethylene glycol-treated rats. As a result, compared with control group, higher urinary and serum concentrations of vanin-1 were observed in the ethylene glycol-treated group. These results suggest that vanin-1 is a useful and rapid biomarker for renal tubular injury induced by organic solvents.

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 ∼09:00u2009h. There were significant correlations between (1) waist circumference and mRNA level of PER1 (ru2009=−0.43), (2) plasma glucose concentration and PER2 (ru2009=−0.50), (3) ethanol consumption and BMAL1 (ru2009=−0.43), and (4) serum γ-GTP concentration (a sensitive marker of alcohol consumption) and PER2 (ru2009=−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)