Background: Mitochondrial dysfunction is strongly associated with several kidney diseases. However, no studies have evaluated the potential renal hazards of serum mitochondria-inhibiting substance (MIS) and aryl hydrocarbon receptor ligand (AhRL) levels. Methods: We used serum level of MIS and AhRL and clinical renal outcomes from 1,511 participants of a prospective community-based cohort in Ansung. MIS was evaluated based on intracellular adenosine triphosphate (MIS-ATP) or reactive oxygen species (MIS-ROS) generation measured using cell-based assays. Results: During a mean 6.9-year follow-up, 84 participants (5.6%) developed a rapid decline in kidney function. In the lowest quartile group of MIS-ATP, patients were older and had metabolically deleterious parameters. In multivariate logistic regression analysis, higher MIS-ATP was associated with decreased odds for rapid decline: the odds ratio (OR) of 1% increase was 0.977 (95% confidence interval [CI], 0.957 to 0.998; P=0.031), while higher MIS-ROS was marginally associated with increased odds for rapid decline (OR, 1.014; 95% CI, 0.999 to 1.028; P=0.055). However, serum AhRL was not associated with the rapid decline in kidney function. In subgroup analysis, the renal hazard of MIS was particularly evident in people with hypertension and low baseline kidney function. Conclusion Serum MIS was independently associated with a rapid decline in kidney function, while serum AhRL was not. The clinical implication of renal hazard on serum MIS requires further evaluation in future studies.

Mitochondrial deficits or altered expressions of microRNAs are associated with the pathogenesis of various diseases, and microRNA-operated control of mitochondrial activity has been reported. Using a retrovirus-mediated short-hairpin RNA (shRNA) system, we observed that miR-24-mediated H2AX knockdown (H2AX-KD) impaired both mitochondria and the insulin signaling pathway. The overexpression of miR-24 decreased mitochondrial H2AX and disrupted mitochondrial function, as indicated by the ATP content, membrane potential and oxygen consumption. Similar mitochondrial damage was observed in shH2AX-mediated specific H2AX-KD cells. The H2AX-KD reduced the expression levels of mitochondrial transcription factor A (TFAM) and mitochondrial DNA-dependent transcripts. H2AX-KD mitochondria were swollen, and their cristae were destroyed. H2AX-KD also blocked the import of precursor proteins into mitochondria and the insulin-stimulated phosphorylation of IRS-1 (Y632) and Akt (S473 and T308). The rescue of H2AX, but not the nuclear form of ΔC24-H2AX, restored all features of miR-24- or shH2AX-mediated impairment of mitochondria. Hepatic miR-24 levels were significantly increased in db/db and ob/ob mice. A strong feedback loop may be present among miR-24, H2AX, mitochondria and the insulin signaling pathway. Our findings suggest that H2AX-targeting miR-24 may be a novel negative regulator of mitochondrial function and is implicated in the pathogenesis of insulin resistance.
Adenosine Triphosphate ; Animals ; Insulin Resistance ; Insulin* ; Membrane Potentials ; Mice ; MicroRNAs ; Mitochondria* ; Oxygen Consumption ; Phosphorylation ; RNA ; Transcription Factors

Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.
ARNTL Transcription Factors/physiology ; Animals ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism/physiology ; CLOCK Proteins/physiology ; Cholesterol/blood ; *Circadian Rhythm ; E-Box Elements ; Exons ; *Gene Expression Regulation ; Hep G2 Cells ; Homeodomain Proteins/*genetics/metabolism/physiology ; Homeostasis ; Humans ; Liver/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; *Promoter Regions, Genetic ; Receptors, LDL/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism/physiology ; Transcription, Genetic

Hypertension and anemia may be causes of left ventricular hypertrophy (LVH) in uremia but the molecular mechanism is not known. Uremia was induced in male Spraugue Dawley rats by 5/6 nephrectomy. The following groups of rats were studied for 6 weeks; uremic rats (U) fed ad. lib., control rats (C) pair-fed with U, U rats given hydralazine (100 mg/kg/day) (UH), U rats given erythropoietin (48U/kg/week, i.p.) (UE). Both diastolic and mean arterial pressures are higher (P<0.01) in U and UE compared with C whereas both pressures in UH were normalized. Hemoglobin in U was lower than in C, and was normalized in UE. U, UH and UE had higher heart weight/body weight ratios (HW/BW) as well as left ventricular weight/body weight ratios (LV/BW) compared with C (P<0.01). Compared with U, UH has lower HW/BW and LV/BW (P <0.05) and UE has normal HW/BW but lower LV/BW than U (P<0.05). To see if the gene expression in uremic LVH is similar to that described in pressure overload LVH in which mRNA levels of angiotensin converting enzyme (ACE), transforming growth factor-beta1 (TGF-beta1), atrial natriuretic factors (ANF) and skeletal alpha-actin were increased, we measured these mRNA levels by Northern analysis. TGF-beta, ACE and alpha-actin mRNA levels were not changed in all 4 groups. ANF mRNA in U and UE was increased 3 fold over C, and normalized in UH. Treatment of anemia with erythropoietin improved uremic LVH but did not change ANF mRNA; whereas treatment of hypertension with hydralazine normalized ANF mRNA but did not completely correct uremic LVH. Thus, gene expression in uremic LVH is distinct from that in pressure- overload LVH, suggesting that other unidentified factor(s) might be involved in uremic LVH.
Actins/genetics/metabolism ; Anemia/*complications/drug therapy/metabolism ; Animals ; Atrial Natriuretic Factor/genetics/metabolism ; Erythropoietin/pharmacology/therapeutic use ; *Gene Expression ; Heart Ventricles/chemistry/drug effects/pathology ; Hydralazine/pharmacology/therapeutic use ; Hypertension/*complications/drug therapy/metabolism ; Hypertrophy, Left Ventricular/etiology/*genetics/metabolism ; Male ; Peptidyl-Dipeptidase A/genetics/metabolism ; RNA, Messenger/analysis/metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta/genetics/metabolism ; Uremia/etiology/*genetics/metabolism

Insulin-like growth factor (IGF)/IGF binding protein (IGFBP) abnormalities may be important in the pathogenesis of growth failure in chronic renal failure (CRF). We induced experimental CRF by 5/6 nephrectomy in Sprague Dawley rats (100 g) and observed for 2 weeks comparing with sham-operated pair-fed control rats (Sham- C). CRF rats gained 30% less height than Sham- C rats (P<0.01). Serum IGFBP profiles by Western ligand blot revealed that IGFBP4 was elevated two fold in CRF rats (P<0.01 vs. Sham-C). However, IGFBP4 mRNA levels in liver or skeletal muscle were not different in two groups. To determine if the increase of serum IGFBP4 in CRF retarded the growth of cartilage, epiphyseal chondrocytes were isolated from CRF or control rats and cultured in the presence of control or CRF rat sera. Incubation with 10% CRF serum reduced proliferations of normal chondrocytes and L6 rat skeletal muscle cells. In contrast, 10% CRF serum did not inhibit the growth of CRF chondrocytes. Rat sera from two groups were separated into two different fractions, high (>10 kDa, containing IGFBPs) and low (<10 kDa, containing free IGF) molecular weight fractions using a gel filtration column. Both fractions obtained from CRF sera decreased the growth of control chondrocytes up to 40% compared with those from control sera. We suggest that the pathogenesis of growth failure in CRF may be involved in the increase of circulating IGFBP4 as well as the unidentified small molecular weight uremic serum factors which block the growth of chondrocytes in growth plate.
Animals ; Cell Proliferation ; Cells, Cultured ; Chondrocytes/*cytology/metabolism ; Insulin-Like Growth Factor Binding Protein 4/analysis/*blood/genetics ; Kidney Failure, Chronic/*blood/metabolism/physiopathology ; Liver/chemistry ; Male ; Muscle, Skeletal/chemistry ; RNA, Messenger/analysis/metabolism ; Rats ; Rats, Sprague-Dawley ; Somatomedins/analysis/metabolism

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.
Animals ; Bile Acids and Salts/metabolism ; Cholesterol/*metabolism ; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism ; Forkhead Transcription Factors/genetics/*metabolism ; Gene Expression Regulation/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Humans ; Insulin/pharmacology ; Lipid Metabolism/drug effects ; Liver/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins/genetics/*metabolism ; Protein Binding/drug effects/genetics ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Receptors, Cytoplasmic and Nuclear/genetics/metabolism ; Sequence Deletion/genetics ; Signal Transduction/drug effects/genetics ; Transcriptional Activation/drug effects/genetics

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.
Animals ; Bile Acids and Salts/metabolism ; Cholesterol/*metabolism ; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism ; Forkhead Transcription Factors/genetics/*metabolism ; Gene Expression Regulation/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Humans ; Insulin/pharmacology ; Lipid Metabolism/drug effects ; Liver/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins/genetics/*metabolism ; Protein Binding/drug effects/genetics ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Receptors, Cytoplasmic and Nuclear/genetics/metabolism ; Sequence Deletion/genetics ; Signal Transduction/drug effects/genetics ; Transcriptional Activation/drug effects/genetics

BACKGROUND/OBJECTIVES: The diet is an important route of exposure to endocrinedisrupting chemicals (EDCs). However, few studies have investigated the association between dietary intake and EDC exposure levels among Koreans. In an earlier study, we showed that the bioactivity of serum aryl hydrocarbon receptor ligands (AhRLs) could be a surrogate biomarker to indicate exposure to EDCs and that they inhibit mitochondrial function. We also found that the mitochondria-inhibiting substances (MIS) in serum ascertained by intracellular adenosine triphosphate (MIS-ATP) and reactive oxygen species (MIS-ROS) levels could be biomarkers of exposure to EDCs, as they showed a strong correlation with AhRL and the levels of EDCs in the blood. Here, we investigated the association between the consumption of specific foods and surrogate serum biomarkers for EDCs, namely AhRL, MIS-ATP, and MIS-ROS, among middle-aged Korean adults. SUBJECTS/METHODS: A total of 1,466 participants aged 45–76 yrs from the Ansung cohort of the Korean Genome and Epidemiology Study were included. Food consumption, including that of meat, fish, vegetables, and fruits, was measured using a semiquantitative food frequency questionnaire. RESULTS: Fish intake was positively associated with AhRL (β = 0.0035, P = 0.0166), whereas cruciferous vegetable intake was negatively associated with AhRL (β = −0.0007, P = 0.0488).Cruciferous vegetable intake was positively associated with the MIS-ATP levels (β = 0.0051, P = 0.0420). A higher intake of fish was significantly associated with an increased risk of high AhRL (tertile: odds ratio [OR], 1.49; 95% confidence intervals (CIs), 1.08–2.06; P for trend = 0.0305). In addition, the second-highest tertile of cruciferous vegetable intake had lower odds of high AhRL than the lowest tertile (OR, 0.73; 95% CIs, 0.54–0.97), although no significant linear trend was observed. CONCLUSION Consumption of different types of foods may be differentially associated with EDC exposure in middle-aged Korean adults.

BACKGROUND/OBJECTIVES: The diet is an important route of exposure to endocrinedisrupting chemicals (EDCs). However, few studies have investigated the association between dietary intake and EDC exposure levels among Koreans. In an earlier study, we showed that the bioactivity of serum aryl hydrocarbon receptor ligands (AhRLs) could be a surrogate biomarker to indicate exposure to EDCs and that they inhibit mitochondrial function. We also found that the mitochondria-inhibiting substances (MIS) in serum ascertained by intracellular adenosine triphosphate (MIS-ATP) and reactive oxygen species (MIS-ROS) levels could be biomarkers of exposure to EDCs, as they showed a strong correlation with AhRL and the levels of EDCs in the blood. Here, we investigated the association between the consumption of specific foods and surrogate serum biomarkers for EDCs, namely AhRL, MIS-ATP, and MIS-ROS, among middle-aged Korean adults. SUBJECTS/METHODS: A total of 1,466 participants aged 45–76 yrs from the Ansung cohort of the Korean Genome and Epidemiology Study were included. Food consumption, including that of meat, fish, vegetables, and fruits, was measured using a semiquantitative food frequency questionnaire. RESULTS: Fish intake was positively associated with AhRL (β = 0.0035, P = 0.0166), whereas cruciferous vegetable intake was negatively associated with AhRL (β = −0.0007, P = 0.0488).Cruciferous vegetable intake was positively associated with the MIS-ATP levels (β = 0.0051, P = 0.0420). A higher intake of fish was significantly associated with an increased risk of high AhRL (tertile: odds ratio [OR], 1.49; 95% confidence intervals (CIs), 1.08–2.06; P for trend = 0.0305). In addition, the second-highest tertile of cruciferous vegetable intake had lower odds of high AhRL than the lowest tertile (OR, 0.73; 95% CIs, 0.54–0.97), although no significant linear trend was observed. CONCLUSION Consumption of different types of foods may be differentially associated with EDC exposure in middle-aged Korean adults.

BACKGROUND/OBJECTIVES: The diet is an important route of exposure to endocrinedisrupting chemicals (EDCs). However, few studies have investigated the association between dietary intake and EDC exposure levels among Koreans. In an earlier study, we showed that the bioactivity of serum aryl hydrocarbon receptor ligands (AhRLs) could be a surrogate biomarker to indicate exposure to EDCs and that they inhibit mitochondrial function. We also found that the mitochondria-inhibiting substances (MIS) in serum ascertained by intracellular adenosine triphosphate (MIS-ATP) and reactive oxygen species (MIS-ROS) levels could be biomarkers of exposure to EDCs, as they showed a strong correlation with AhRL and the levels of EDCs in the blood. Here, we investigated the association between the consumption of specific foods and surrogate serum biomarkers for EDCs, namely AhRL, MIS-ATP, and MIS-ROS, among middle-aged Korean adults. SUBJECTS/METHODS: A total of 1,466 participants aged 45–76 yrs from the Ansung cohort of the Korean Genome and Epidemiology Study were included. Food consumption, including that of meat, fish, vegetables, and fruits, was measured using a semiquantitative food frequency questionnaire. RESULTS: Fish intake was positively associated with AhRL (β = 0.0035, P = 0.0166), whereas cruciferous vegetable intake was negatively associated with AhRL (β = −0.0007, P = 0.0488).Cruciferous vegetable intake was positively associated with the MIS-ATP levels (β = 0.0051, P = 0.0420). A higher intake of fish was significantly associated with an increased risk of high AhRL (tertile: odds ratio [OR], 1.49; 95% confidence intervals (CIs), 1.08–2.06; P for trend = 0.0305). In addition, the second-highest tertile of cruciferous vegetable intake had lower odds of high AhRL than the lowest tertile (OR, 0.73; 95% CIs, 0.54–0.97), although no significant linear trend was observed. CONCLUSION Consumption of different types of foods may be differentially associated with EDC exposure in middle-aged Korean adults.