Diagnostic and Prognostic Significance of Serum Levels of SeP (Selenoprotein P) in Patients With Pulmonary Hypertension.

OBJECTIVE: Despite the recent progress in upfront combination therapy for pulmonary arterial hypertension (PAH), useful biomarkers for the disorder still remain to be developed. SeP (Selenoprotein P) is a glycoprotein secreted from various kinds of cells including pulmonary artery smooth muscle cells to maintain cellular metabolism. We have recently demonstrated that SeP production from pulmonary artery smooth muscle cells is upregulated and plays crucial roles in the pathogenesis of PAH. However, it remains to be elucidated whether serum SeP levels could be a useful biomarker for PAH. Approach and Results: We measured serum SeP levels and evaluated their prognostic impacts in 65 consecutive patients with PAH and 20 controls during follow-up (mean, 1520 days; interquartile range, 1393-1804 days). Serum SeP levels were measured using a newly developed sol particle homogeneous immunoassay. The patients with PAH showed significantly higher serum SeP levels compared with controls. Higher SeP levels (cutoff point, 3.47 mg/L) were associated with the outcome (composite end point of all-cause death and lung transplantation) in patients with PAH (hazard ratio, 4.85 [1.42-16.6]; P<0.01). Importantly, we found that the absolute change in SeP of patients with PAH (ΔSeP) in response to the initiation of PAH-specific therapy significantly correlated with the absolute change in mean pulmonary artery pressure, pulmonary vascular resistance (ΔPVR), and cardiac index (ΔCI; R=0.78, 0.76, and -0.71 respectively, all P<0.0001). Moreover, increase in ΔSeP during the follow-up predicted poor outcome of PAH. CONCLUSIONS: Serum SeP is a novel biomarker for diagnosis and assessment of treatment efficacy and long-term prognosis in patients with PAH.

Circulating Concentrations of Insulin Resistance-Associated Hepatokines, Selenoprotein P and Leukocyte Cell-Derived Chemotaxin 2, during an Oral Glucose Tolerance Test in Humans.

A hepatokine is a collective term for liver-derived secretory factors whose previously-unrecognized functions have been recently elucidated. We have rediscovered selenoprotein P (SeP) and leukocyte cell-derived chemotaxin 2 (LECT2) as hepatokines that are involved in the development of insulin resistance and hyperglycemia. The aim of this study was to determine whether and, if so, how oral glucose loading alters the two hepatokines in humans. We measured concentrations of serum SeP and plasma LECT2 during 75 g oral glucose tolerance test (OGTT) (n = 20) in people with various degrees of glucose tolerance. In OGTT, concentrations of both serum SeP and plasma LECT2 decreased at 120 min compared with the baseline values, irrespective of the severity of glucose intolerance. Decrement of serum SeP during OGTT showed no correlations to the clinical parameters associated with insulin resistance or insulin secretion. In multiple stepwise regression analyses, plasma cortisol was selected as the variable to explain the changes in plasma concentrations of LECT2. The current data reveal the acute inhibitory actions of oral intake of glucose on circulating SeP and LECT2 in humans, irrespective of the severity of glucose intolerance. This study suggests that circulating SeP is regulated by the unknown clinical factors other than insulin and glucose during OGTT.

Identification of hepatokines involved in pathology of type 2 diabetes and obesity.

Many researchers pay attention to novel secretory factors, such as adipokines or osteokines, secreted by the tissues that were not formerly recognized as classical endocrine organs. The liver also contributes to the onset of various kinds of pathologies of type 2 diabetes and obesity by producing and releasing secretory proteins "hepatokines." By using the information of gene expression in human livers, we rediscovered selenoprotein P (SeP) and leukocyte cell-derived chemotaxin 2 (LECT2) as hepatokines involved in the onset of glucose intolerance. SeP was previously recognized as a selenium transport protein, but we revealed that SeP causes insulin resistance in the muscle and liver. SeP also reduces VEGF signal transduction in vascular endothelial cells, contributing the impaired angiogenesis in diabetes. Importantly, SeP impairs health-promoting effects of exercise training by suppressing reactive oxygen species (ROS)/adenosine monophosphate-dependent protein kinase (AMPK) pathway in the skeletal muscle through its receptor low-density lipoprotein receptor-related protein 1 (LRP1). LECT2, previously-reported as a neutrophil chemotactic protein, promotes skeletal muscle insulin resistance in obesity. Further studies are necessary to develop new diagnostic or therapeutic procedures targeting hepatokines to combat type 2 diabetes or obesity.

Eicosapentaenoic acid down-regulates expression of the selenoprotein P gene by inhibiting SREBP-1c protein independently of the AMP-activated protein kinase pathway in H4IIEC3 hepatocytes.

Selenoprotein P (encoded by SELENOP in humans, Selenop in rat), a liver-derived secretory protein, induces resistance to insulin and vascular endothelial growth factor (VEGF) in type 2 diabetes. Suppression of selenoprotein P may provide a novel therapeutic approach to treating type 2 diabetes; however, few drugs inhibiting SELENOP expression in hepatocytes have been identified. The present findings demonstrate that eicosapentaenoic acid (EPA) suppresses SELENOP expression by inactivating sterol regulatory element-binding protein-1c (SREBP-1c, encoded by Srebf1 in rat) in H4IIEC3 hepatocytes. Treatment with EPA caused concentration- and time-dependent reduction in SELENOP promoter activity. EPA activated AMP-activated protein kinase (AMPK); however, the inhibitory effect of EPA on SELENOP promoter activity was not canceled with an AMPK inhibitor compound C and dominant-negative AMPK transfection. Deletion mutant promoter assays and computational analysis of transcription factor-binding sites conserved among the species resulted in identification of a sterol regulatory element (SRE)-like site in the SELENOP promoter. A chromatin immunoprecipitation (ChIP) assay revealed that EPA decreases binding of SREBP-1c to the SELENOP promoter. Knockdown of Srebf1 resulted in a significant down-regulation of Selenop expression. Conversely, SREBP-1c overexpression inhibited the suppressive effect of EPA. These data provide a novel mechanism of action for EPA involving improvement of systemic insulin sensitivity through the regulation of selenoprotein P production independently of the AMPK pathway and suggest an additional approach to developing anti-diabetic drugs.

Comparison of Human Selenoprotein P Determinants in Serum between Our Original Methods and Commercially Available Kits.

Selenoprotein P (SeP) is a selenium (Se)-rich extracellular protein. SeP is identified as a hepatokine, causing insulin resistance in type 2 diabetes. Thus, the measurement of SeP in serum has received much attention, and several enzyme-linked immunosorbent assay (ELISA) kits for SeP determination are now commercially available. In the present study, we determined the serum SeP levels by our original ELISA and sol particle homogeneous immunoassay (SPIA) methods and also by commercially available kits, and these determinants were compared. We found a kit-dependent correlation of the determinants with our methods. These results suggest that the selection of kit is critical for comparison with our previous reports and for discussing the relationship between the serum SeP levels and disease condition.

Endogenous Selenoprotein P, a Liver-Derived Secretory Protein, Mediates Myocardial Ischemia/Reperfusion Injury in Mice.

Selenoprotein P (SeP), a liver-derived secretory protein, functions as a selenium supply protein in the body. SeP has been reported to be associated with insulin resistance in humans through serial analysis of gene expression. Recently, SeP has been found to inhibit vascular endothelial growth factor-stimulated cell proliferation in human umbilical vein endothelial cells, and impair angiogenesis in a mouse hind limb model. In this study, the role of SeP in ischemia/reperfusion (I/R) injury has been investigated. SeP knockout (KO) and littermate wild-type (WT) mice were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion. The myocardial infarct area/area at risk (IA/AAR), evaluated using Evans blue (EB) and 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in SeP KO mice than in WT mice. The number of terminal de-oxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei was significantly lower in SeP KO mice than in WT mice. In addition, caspase-3 activation was reduced in SeP KO mice compared to that in WT mice. Furthermore, phosphoinositide 3-kinase/Akt and Erk levels were examined for the reperfusion injury salvage kinase (RISK) pathway. Interestingly, SeP KO significantly increased the phosphorylation of IGF-1, Akt, and Erk compared to that in WT mice after I/R. Finally, I/R-induced myocardial IA/AAR was significantly increased in SeP KO mice overexpressing SeP in the liver compared to other SeP KO mice. These results, together, suggest that inhibition of SeP protects the heart from I/R injury through upregulation of the RISK pathway.

Rapid response of the steatosis-sensing hepatokine LECT2 during diet-induced weight cycling in mice.

Dieting often leads to body weight cycling involving repeated weight loss and regain. However, little information is available regarding rapid-response serum markers of overnutrition that predict body weight alterations during weight cycling. Here, we report the rapid response of serum leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine that induces insulin resistance in skeletal muscle, during diet-induced weight cycling in mice. A switch from a high-fat diet (HFD) to a regular diet (RD) in obese mice gradually decreased body weight but rapidly decreased serum LECT2 levels within 10 days. In contrast, a switch from a RD to a HFD rapidly elevated serum LECT2 levels. Serum LECT2 levels showed a positive correlation with liver triglyceride contents but not with adipose tissue weight. This study demonstrates the rapid response of LECT2 preceding body weight alterations during weight cycling in mice and suggests that measurement of serum LECT2 may be clinically useful in the management of obesity.

Development of a Sol Particle Homogeneous Immunoassay for Measuring Full-Length Selenoprotein P in Human Serum.

BACKGROUND: Selenoprotein P (SeP), a selenium-rich extracellular glycoprotein, is the primary selenoprotein in the plasma. SeP plays an important role in the maintenance of selenium levels in the peripheral tissues. We developed a new sol particle homogeneous immunoassay (SPIA) for measuring full-length SeP (FL-SeP) levels in the human serum. METHODS: We used colloidal gold particles coated with two types of anti-SeP monoclonal antibodies, one recognizing the N-terminal side domain of SeP and the other recognizing the C-terminal side domain. RESULTS: The assay range was 0.2-9 mg/l, and the linearity was excellent. The within-day and between-day coefficients of variation ranged from 0.73% to 2.24% and 0.45% to 1.11%, respectively. Serum samples (n = 200) were examined using the newly developed assay system (employing a Model 7070 Hitachi automatic clinical analyzer) and the conventional enzyme-linked immunosorbent assay. These two methods were compared using the Passing-Bablok regression analysis; the resulting regression equation and correlation coefficient were y = 0.940x + 0.165 and r = 0.954, respectively. CONCLUSIONS: Our new SPIA assay is a fully automated homogeneous immunoassay that can be used in conjunction with various commercial analyzers. The assay was sensitive, precise, and suitable for clinical measurement of the FL-SeP in the human serum.

Metformin suppresses expression of the selenoprotein P gene via an AMP-activated kinase (AMPK)/FoxO3a pathway in H4IIEC3 hepatocytes.

Selenoprotein P (SeP; encoded by SEPP1 in humans) is a liver-derived secretory protein that induces insulin resistance in type 2 diabetes. Suppression of SeP might provide a novel therapeutic approach to treating type 2 diabetes, but few drugs that inhibit SEPP1 expression in hepatocytes have been identified to date. The present findings demonstrate that metformin suppresses SEPP1 expression by activating AMP-activated kinase (AMPK) and subsequently inactivating FoxO3a in H4IIEC3 hepatocytes. Treatment with metformin reduced SEPP1 promoter activity in a concentration- and time-dependent manner; this effect was cancelled by co-administration of an AMPK inhibitor. Metformin also suppressed Sepp1 gene expression in the liver of mice. Computational analysis of transcription factor binding sites conserved among the species resulted in identification of the FoxO-binding site in the metformin-response element of the SEPP1 promoter. A luciferase reporter assay showed that metformin suppresses Forkhead-response element activity, and a ChIP assay revealed that metformin decreases binding of FoxO3a, a direct target of AMPK, to the SEPP1 promoter. Transfection with siRNAs for Foxo3a, but not for Foxo1, cancelled metformin-induced luciferase activity suppression of the metformin-response element of the SEPP1 promoter. The overexpression of FoxO3a stimulated SEPP1 promoter activity and rescued the suppressive effect of metformin. Metformin did not affect FoxO3a expression, but it increased its phosphorylation and decreased its nuclear localization. These data provide a novel mechanism of action for metformin involving improvement of systemic insulin sensitivity through the regulation of SeP production and suggest an additional approach to the development of anti-diabetic drugs.

Characteristics of hepatic fatty acid compositions in patients with nonalcoholic steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and lipid metabolism. Recent studies have suggested that the quality of fat accumulated in the liver is associated with the development of nonalcoholic steatohepatitis (NASH). In this study, we investigated the fatty acid composition in liver tissue and its association with the pathology in NAFLD patients. METHODS: One hundred and three patients diagnosed with NAFLD [simple steatosis (SS): 63, NASH: 40] were examined and their hepatic fatty acids were measured using gas chromatography. In addition, relationships between the composition and composition ratios of various fatty acids and patient backgrounds, laboratory test values, histology of the liver, and expression of fat metabolism-related enzymes were investigated. RESULTS: The C16:1n7 content, the C16:1n7/C16:0 and C18:1n9/C18:0 ratios were increased and the C18:0/C16:0 ratio was decreased in the NASH group. The C18:0/C16:0 and C18:1n9/C18:0 ratios were associated with the steatosis score in liver tissue, and the C16:1n7/C16:0 ratio was associated with the lobular inflammation score. The expressions levels of genes: SCD1, ELOVL6, SREBP1c, FAS and PPARγ were enhanced in the NASH group. In multivariate analysis, the C18:0/C16:0 ratio was the most important factor that was correlated with the steatosis score. In contrast, the C16:1n7/C16:0 ratio was correlated with lobular inflammation. CONCLUSION: The fatty acid composition in liver tissue and expression of genes related to fatty acid metabolism were different between the SS and NASH groups, suggesting that the acceleration of fatty acid metabolism is deeply involved in pathogenesis of NASH.

The effects of ezetimibe on non-alcoholic fatty liver disease and glucose metabolism: a randomised controlled trial.

AIMS/HYPOTHESIS: The cholesterol absorption inhibitor ezetimibe has been shown to ameliorate non-alcoholic fatty liver disease (NAFLD) pathology in a single-armed clinical study and in experimental animal models. In this study, we investigated the efficacy of ezetimibe on NAFLD pathology in an open-label randomised controlled clinical trial. METHODS: We had planned to enrol 80 patients in the trial, as we had estimated that, with this sample size, the study would have 90% power. The study intervention and enrolment were discontinued because of the higher proportion of adverse events (significant elevation in HbA(1c)) in the ezetimibe group than in the control group. Thirty-two patients with NAFLD were enrolled and randomised (allocation by computer program). Ezetimibe (10 mg/day) was given to 17 patients with NAFLD for 6 months. The primary endpoint was change in serum aminotransferase level. Secondary outcomes were change in liver histology (12 control and 16 ezetimibe patients), insulin sensitivity including a hyperinsulinaemic-euglycaemic clamp study (ten control and 13 ezetimibe patients) and hepatic fatty acid composition (six control and nine ezetimibe patients). Hepatic gene expression profiling was completed in 15 patients using an Affymetrix gene chip. Patients and the physician in charge knew to which group the patient had been allocated, but people carrying out measurements or examinations were blinded to group. RESULTS: Serum total cholesterol was significantly decreased in the ezetimibe group. The fibrosis stage and ballooning score were also significantly improved with ezetimibe treatment. However, ezetimibe treatment significantly increased HbA1c and was associated with a significant increase in hepatic long-chain fatty acids. Hepatic gene expression analysis showed coordinate downregulation of genes involved in skeletal muscle development and cell adhesion molecules in the ezetimibe treatment group, suggesting a suppression of stellate cell development into myofibroblasts. Genes involved in the L-carnitine pathway were coordinately downregulated by ezetimibe treatment and those in the steroid metabolism pathway upregulated, suggestive of impaired oxidation of long-chain fatty acids. CONCLUSIONS/INTERPRETATION: Ezetimibe improved hepatic fibrosis but increased hepatic long-chain fatty acids and HbA1c in patients with NAFLD. These findings shed light on previously unrecognised actions of ezetimibe that should be examined further in future studies. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN) Clinical Trials Registry UMIN000005250. FUNDING: The study was funded by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and research grants from MSD.

Selenoprotein P as a diabetes-associated hepatokine that impairs angiogenesis by inducing VEGF resistance in vascular endothelial cells.

AIMS/HYPOTHESIS: Impaired angiogenesis induced by vascular endothelial growth factor (VEGF) resistance is a hallmark of vascular complications in type 2 diabetes; however, its molecular mechanism is not fully understood. We have previously identified selenoprotein P (SeP, encoded by the SEPP1 gene in humans) as a liver-derived secretory protein that induces insulin resistance. Levels of serum SeP and hepatic expression of SEPP1 are elevated in type 2 diabetes. Here, we investigated the effects of SeP on VEGF signalling and angiogenesis. METHODS: We assessed the action of glucose on Sepp1 expression in cultured hepatocytes. We examined the actions of SeP on VEGF signalling and VEGF-induced angiogenesis in HUVECs. We assessed wound healing in mice with hepatic SeP overexpression or SeP deletion. The blood flow recovery after ischaemia was also examined by using hindlimb ischaemia model with Sepp1-heterozygous-knockout mice. RESULTS: Treatment with glucose increased gene expression and transcriptional activity for Sepp1 in H4IIEC hepatocytes. Physiological concentrations of SeP inhibited VEGF-stimulated cell proliferation, tubule formation and migration in HUVECs. SeP suppressed VEGF-induced reactive oxygen species (ROS) generation and phosphorylation of VEGF receptor 2 (VEGFR2) and extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs. Wound closure was impaired in the mice overexpressing Sepp1, whereas it was improved in SeP (-/-)mice. SeP (+/-)mice showed an increase in blood flow recovery and vascular endothelial cells after hindlimb ischaemia. CONCLUSIONS/INTERPRETATION: The hepatokine SeP may be a novel therapeutic target for impaired angiogenesis in type 2 diabetes.

Pharmacokinetics and pharmacodynamics of insulin aspart in patients with type 2 diabetes: assessment using a meal tolerance test under clinical conditions.

1. Few studies have evaluated the pharmacokinetics of rapid-acting insulin analogues in patients with Type 2 diabetes, especially under clinical conditions. The aim of the present study was to assess both the pharmacokinetics and pharmacodynamics of insulin aspart in Type 2 diabetic patients who were being treated with the analogue alone. 2. Meal tolerance tests with and without self-injection of a customary dose of insulin aspart (0.05-0.22 U/kg) were conducted in 20 patients in a randomized cross-over study. 3. The dose of insulin aspart (per bodyweight) was significantly correlated with both the maximum concentration (r(2) = 0.59; P < 0.01) and area under the concentration-time curve for insulin aspart (r(2) = 0.53; P < 0.01). However, the time to maximum concentration (T(max)), which varied widely from < 60 to ≥ 120 min, was not associated with either dosage (r(2) = 0.02; P = 0.51) or body mass index (r(2) = 0.02; P = 0.57). Injection of insulin aspart exacerbated delayed hyperinsulinaemia after meal loading, mainly in patients with T(max) ≥ 120 min. With regard to pharmacodynamics, insulin aspart had favourable effects on postprandial hyperglycaemia, hyperglucagonaemia and hyperlipidaemia. 4. The T(max) for this insulin analogue differed greatly between individuals and delayed hyperinsulinaemia was particularly exacerbated in patients with higher T(max) values. Identification of the factors contributing to interindividual variation in the absorption lag time is essential for improving the efficacy and safety of insulin aspart.

Fatty liver as a consequence and cause of insulin resistance: lessons from type 2 diabetic liver.

Obesity is less common in the Asian population, but Asian people may be susceptible to obesity-associated metabolic dysregulation. Accumulating evidence suggests that insulin resistance is closely associated with ectopic fat accumulation in the liver. Whether this correlation is due to a causal relationship between the conditions has long been the subject of debate. Insulin resistance and type 2 diabetes affects liver pathology, typically leading to nonalcoholic fatty liver disease (NAFLD) by dynamically altering the hepatic genes involved in glucose and lipid metabolism. Conversely, how overnutrition induces hepatic insulin resistance has been studied intensively, and has been shown to involve excessive energy flux into mitochondria, toxic lipids, reactive oxygen species, and hepatokines. In this review, we focus on NAFLD both as a consequence and as a cause of insulin resistance through lessons learned from the liver of patients with type 2 diabetes.

Duodenal adenocarcinoma with neuroendocrine features in a patient with acromegaly and thyroid papillary adenocarcinoma: a unique combination of endocrine neoplasia.

A 67-year-old woman with familial clustering of thyroid papillary adenocarcinoma was diagnosed with acromegaly due to pituitary macroadenoma. She had multiple skin vegetations, but had no parathyroid and pancreas diseases. Before transsphenoidal surgery, she was further diagnosed as having a duodenal tumor and multiple hypervascular liver nodules. Biopsy specimens from the duodenal tumor and liver nodules were diagnosed histologically as moderately differentiated adenocarcinoma. Immunohistochemically, the tumor cells were positive for chromogranin, synaptophysin and somatostatin receptor 2a, suggestive for neuroendocrine features. After surgery, the patient was not in biochemical remission, and octreotide treatment was initiated. The duodenal cancer was treated with chemotherapy (neoadjuvant cisplatin and S-1). After 24 months, the patient's insulin-like growth factor I level had been normalized, and her liver tumors had not progressed macroscopically. This is a rare case of acromegaly associated with multiple endocrine tumors, not being categorized as conventional multiple endocrine neoplasia. Octreotide treatment might have had beneficial effects on our patient's duodenal adenocarcinoma and liver metastases, both directly via SSTR2a and indirectly via GH suppression, thereby contributing to their slow progression.

[Novel strategies that target hepatokines for treatment of type 2 diabetes].

The liver may maintain systemic homeostasis by releasing secretory proteins into the blood stream. In fact, comprehensive gene expression analysis revealed that the liver in humans expresses various kinds of genes encoding secretory proteins. Recently, function-unknown liver-derived secretory proteins have been termed as hepatokines. Using comprehensive gene expression analyses, we have identified selenoprotein P(SeP) as a novel hepatokine that elevates plasma glucose levels by inducing insulin resistance. RNA interference-mediated knockdown of SeP in the liver improved insulin resistance and hyperglycemia in type 2 diabetic mice, suggesting that SeP is a novel therapeutic target for type 2 diabetes. Further functional characterization of hepatokines might provide novel diagnostic and therapeutic targets for the other diseases except diabetes.

Plasma half-life and tissue distribution of leukocyte cell-derived chemotaxin 2 in mice.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine that causes skeletal muscle insulin resistance. The circulating levels of LECT2 are a possible biomarker that can predict weight cycling because they reflect liver fat and precede the onset of weight loss or gain. Herein, to clarify the dynamics of this rapid change in serum LECT2 levels, we investigated the in vivo kinetics of LECT2, including its plasma half-life and tissue distribution, by injecting 125I-labelled LECT2 into ICR mice and radioactivity tracing. The injected LECT2 was eliminated from the bloodstream within 10 min (approximate half-life, 5 min). In the kidneys, the radioactivity accumulated within 10 min after injection and declined thereafter. Conversely, the radioactivity in urine increased after 30 min of injection, indicating that LECT2 is mainly excreted by the kidneys into the urine. Finally, LECT2 accumulated in the skeletal muscle and liver until 30 min and 2 min after injection, respectively. LECT2 accumulation was not observed in the adipose tissue. These findings are in agreement with LECT2 action on the skeletal muscle. The present study indicates that LECT2 is a rapid-turnover protein, which renders the circulating level of LECT2 a useful rapid-response biomarker to predict body weight alterations.

The hepatic circadian clock is preserved in a lipid-induced mouse model of non-alcoholic steatohepatitis.

Recent studies have correlated metabolic diseases, such as metabolic syndrome and non-alcoholic fatty liver disease, with the circadian clock. However, whether such metabolic changes per se affect the circadian clock remains controversial. To address this, we investigated the daily mRNA expression profiles of clock genes in the liver of a dietary mouse model of non-alcoholic steatohepatitis (NASH) using a custom-made, high-precision DNA chip. C57BL/6J mice fed an atherogenic diet for 5 weeks developed hypercholesterolemia, oxidative stress, and NASH. DNA chip analyses revealed that the atherogenic diet had a great influence on the mRNA expression of a wide range of genes linked to mitochondrial energy production, redox regulation, and carbohydrate and lipid metabolism. However, the rhythmic mRNA expression of the clock genes in the liver remained intact. Most of the circadianly expressed genes also showed 24-h rhythmicity. These findings suggest that the biological clock is protected against such a metabolic derangement as NASH.