Activation of the IKK2/NF-κB pathway in VSMCs inhibits calcified vascular stiffness in CKD

IKK2/NF-κB pathway-mediated inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2/NF-κB pathway in medial calcification remains to be elucidated. In this study, we found that chronic kidney disease (CKD) induces inflammatory pathways through the local activation of the IKK2/NF-κB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2/NF-κB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF-κB by SMC-specific IκBα deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2/NF-κB pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF-κB reduced CKD-dependent vascular cell death. In addition, increased calcification of extracellular vesicles through the inhibition of the IKK2/NF-κB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2/NF-κB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.


All-trans retinoic acid induces lipophagy through the activation of the AMPK-Beclin1 signaling pathway and reduces Rubicon expression in adipocytes.

Lipophagy is defined as a lipolysis pathway that degrades lipid droplet (LD) via autophagy. All-trans retinoic acid (atRA), a metabolite of vitamin A, stimulates lipolysis through hormone-sensitive lipase and β-oxidation. However, the regulation of lipolysis by atRA-induced autophagy in adipocytes remains unclear. In this study, we investigated the effect of atRA on autophagy in epididymal fat of mice and the molecular mechanisms of autophagy in 3T3-L1 adipocytes. Western blotting showed that atRA decreased the expression of p62, a cargo receptor for autophagic degradation, and increased the expression of the lipidated LC3B (LC3B-II), an autophagy marker, in epididymal fat. Next, we confirmed that atRA increased autophagic flux in differentiated 3T3-L1 cells using the GFP-LC3-RFP-LC3ΔG probe. Immunofluorescent staining revealed that the colocalization of LC3B with perilipin increased in differentiated 3T3-L1 cells treated with atRA. The knockdown of Atg5, an essential gene in autophagy induction, partly suppressed the atRA-induced release of non-esterified fatty acid (NEFA) from LDs in differentiated 3T3-L1 cells. atRA time-dependently elicited the phosphorylation of AMPK and Beclin1, autophagy-inducing factors, in mature 3T3-L1 adipocytes. Inversely, atRA decreased the protein expression of Rubicon, an autophagy repressor, in differentiated 3T3-L1 cells and epididymal fat. Interestingly, the expression of ALDH1A1, atRA-synthesizing enzymes, increased in epididymal fat with decreased protein expression of Rubicon in aged mice. These results suggest that atRA may partially induce lipolysis through lipophagy by activating the AMPK-Beclin1 signaling pathway in the adipocytes and increased atRA levels may contribute to decreased Rubicon expression in the epididymal fat of aged mice. (248/250 words).


Elevated luminal inorganic phosphate suppresses intestinal Zn absorption in 5/6 nephrectomized rats.

binder might be useful for the treatment of hypozincemia and hyperphosphatemia.

American Journal of Physiology, Renal Physiology


Investigation of Amino Acid and Fatty Acid Profiles of Japanese Diets Using the Food Exchange Lists for Diabetes Diet.

Dietary Reference Intakes for Japanese provide target values for proteins, fats, and carbohydrates. However, they do not provide information on reference values for amino acids (AAs) and fatty acids (FAs), which determine the quality of foods in detail. Therefore, we evaluated AAs and FAs using the Food Exchange Lists-Dietary Guidance for Persons with Diabetes (in Japanese) Utilization, Second Edition Sample Menus and Practice (FELD) as an ideal Japanese diet. Based on FELD, 15 different daily meal patterns were employed with combinations of three levels of carbohydrates %energy (high carbohydrate [HC], 60%; middle carbohydrate [MC], 55%; and low carbohydrate [LC], 50%) and five levels of energy (1,200-2,000 kcal). Using the Japanese Food Composition Table 2020 adjusted for 1,000 kcal, 18 AAs, 49 FAs, and calorie densities (CDs, kcal/g) were calculated and compared among the three groups. Dietary AA was rich in glutamic acid, aspartic acid, and leucine; in order, no significant differences were observed among HC, MC, and LC for 18 AAs. Dietary FA was higher for 18:1 total, 16:0, and 18:2 n-6. Moreover, 16:0, 20:0, and 18:1 total in LC and 22:0 and 18:3 n-3 in MC were significantly higher than those in HC. The HC, MC, and LC CD was low at 0.82, 0.84, and 0.93 kcal/g, respectively. No significant differences in 18 AAs and CD were noted among HC, MC, and LC in FELD; however, significant differences were observed in the FA profiles. This study suggests the importance of evaluating diet using AA and FA units.

Journal of Nutritional Science and Vitaminology


Amino acid and fatty acid profiles of the average Japanese diet: Fusion of the National Health and Nutrition Examination Survey and the Food Composition Database


Dietary phosphate disturbs of gut microbiome in mice.

were observed in high phosphate diet group. Furthermore, high phosphate diet induced reduction of microbial diversity and decreased mRNA levels of colonic tight junction markers. These results suggest that the excessive intake of dietary phosphate disturbs gut microbiota and affects intestinal barrier function.

Journal of Clinical Biochemistry and Nutrition


Humoral and cellular factors inhibit phosphate-induced vascular calcification during the growth period.

, obvious calcification was observed in the adult aorta but not in the young aorta. Furthermore, culture in high Pi medium increased the mRNA level of tissue-nonspecific alkaline phosphatase (TNAP), which degrades pyrophosphate, only in the adult aorta. Collectively, our findings indicate that the aorta in growing mouse may be resistant to Pi-induced vascular calcification via a mechanism in which high serum fetuin-A levels and suppressed TNAP expression.


Studies on the pathophysiological roles of fat-soluble vitamin and bioactive lipids in mineral and bone disorder.

骨ミネラル代謝異常における脂溶性ビタミンおよび生理活性脂質の病態生理学的役割の研究


25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) induces ectopic calcification.

Vascular calcification is an important pathogenesis related to cardiovascular disease and high mortality rate in chronic kidney disease (CKD) patients. It has been well-known that hyper-phosphatemia induces osteochondrogenic transition of vascular smooth muscle cells (VSMCs) resulting ectopic calcification in aortic media, cardiac valve, and kidney. However, the detailed mechanism of the ectopic calcification has been not clarified yet. Here, we found that the co-localization of CYP27B1 with the calcified lesions of aorta and arteries in kidney of klotho mutant () mice, and then investigated the role of CYP27B1 in the mineralization of the VSMCs. Under high phosphate condition, overexpression of CYP27B1 induced calcification and osteocalcin mRNA expression in the VSMCs. Inversely, siRNA- inhibited high phosphate-induced calcification of the VSMCs. We also found that the accumulated CYP27B1 protein was glycosylated in the kidney of mice. Therefore, overexpression of CYP27B1-N310A and CYP27B1-T439A, which are a mutation for -linked glycosylation site (N310A) and a mutation for -linked glycosylation site (T439A) in CYP27B1, decreased calcium deposition and expression of RUNX2 induced by high phosphate medium in VSMCs compared with wild-type CYP27B1. These results suggest that extra-renal expression of glycosylated CYP27B1 would be required for ectopic calcification of VSMCs under hyperphosphatemia.


Sulforaphane induces lipophagy through the activation of AMPK-mTOR-ULK1 pathway signaling in adipocytes.

Lipophagy, a form of selective autophagy, degrades lipid droplet (LD) in adipose tissue and the liver. The chemotherapeutic isothiocyanate sulforaphane (SFN) contributes to lipolysis through the activation of hormone-sensitive lipase and the browning of white adipocytes. However, the details concerning the regulation of lipolysis in adipocytes by SFN-mediated autophagy remain unclear. In this study, we investigated the effects of SFN on autophagy in the epididymal fat of mice fed a high-fat diet (HFD) or control-fat diet and on the molecular mechanisms of autophagy in differentiated 3T3-L1 cells. Western blotting revealed that the protein expression of lipidated LC3 (LC3-II), an autophagic substrate, was induced after 3T3-L1 adipocytes treatment with SFN. In addition, SFN increased the LC3-II protein expression in the epididymal fat of mice fed an HFD. Immunofluorescence showed that the SFN-induced LC3 expression was co-localized with LDs in 3T3-L1 adipocytes and with perilipin, the most abundant adipocyte-specific protein, in adipocytes of mice fed an HFD. Next, we confirmed that SFN activates autophagy flux in differentiated 3T3-L1 cells using the mCherry-EGFP-LC3 and GFP-LC3-RFP-LC3 probe. Furthermore, we examined the induction mechanisms of autophagy by SFN in 3T3-L1 adipocytes using western blotting. ATG5 knockdown partially blocked the SFN-induced release of fatty acids from LDs in mature 3T3-L1 adipocytes. SFN time-dependently elicited the phosphorylation of AMPK, the dephosphorylation of mTOR, and the phosphorylation of ULK1 in differentiated 3T3-L1 cells. Taken together, these results suggest that SFN may provoke lipophagy through AMPK-mTOR-ULK1 pathway signaling, resulting in partial lipolysis of adipocytes.


All-trans retinoic acid changes muscle fiber type via increasing GADD34 dependent on MAPK signal.

All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response in differentiating leukemic blasts. The downstream transcriptional factor of PERK, a major arm of unfolded protein response, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of gene via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the gene promoter in skeletal muscle. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on mRNA via p-38 MAPK, resulting in the instability of mRNA. Overexpressed GADD34 in C2C12 cells changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation, which changes muscle fiber type.


Identification of Dietary Phytochemicals Capable of Enhancing the Autophagy Flux in HeLa and Caco-2 Human Cell Lines


Phosphatemic Index Is a Novel Evaluation Tool for Dietary Phosphorus Load: A Whole-Foods Approach.


All-trans retinoic acid reduces the transcriptional regulation of intestinal sodium-dependent phosphate co-transporter gene (Npt2b).

Inorganic phosphate (Pi) homeostasis is regulated by intestinal absorption via type II sodium-dependent co-transporter (Npt2b) and by renal reabsorption via Npt2a and Npt2c. Although we previously reported that vitamin A-deficient (VAD) rats had increased urine Pi excretion through the decreased renal expression of Npt2a and Npt2c, the effect of vitamin A on the intestinal Npt2b expression remains unclear. In this study, we investigated the effects of treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, on the Pi absorption and the Npt2b expression in the intestine of VAD rats, as well as and the underlying molecular mechanisms. In VAD rats, the intestinal Pi uptake activity and the expression of Npt2b were increased, but were reduced by the administration of ATRA. The transcriptional activity of reporter plasmid containing the promoter region of the rat Npt2b gene was reduced by ATRA in NIH3T3 cells overexpressing retinoic acid receptor (RAR) and retinoid X receptor (RXR). On the other hand, CCAAT/enhancer-binding proteins (C/EBP) induced transcriptional activity of the Npt2b gene. Knockdown of the C/EBP gene and a mutation analysis of the C/EBP responsible element in the Npt2b gene promoter indicated that C/EBP plays a pivotal role in the regulation of Npt2b gene transcriptional activity by ATRA. EMSA revealed that the RAR/RXR complex inhibits binding of C/EBP to Npt2b gene promoter. Together, these results suggest that ATRA may reduce the intestinal Pi uptake by preventing C/EBP activation of the intestinal Npt2b gene.


Isorhamnetin, a 3'-methoxylated flavonol, enhances the lysosomal proteolysis in J774.1 murine macrophages in a TFEB-independent manner.

Lysosome is the principal organelle for the ultimate degradation of cellular macromolecules, which are delivered through endocytosis, phagocytosis, and autophagy. The lysosomal functions have been found to be impaired by fatty foods and aging, and more importantly, the lysosomal dysfunction in macrophages has been reported as a risk of atherosclerosis development. In this study, we searched for dietary polyphenols which possess the activity for enhancing the lysosomal degradation in J774.1, a murine macrophage-like cell line. Screening test utilizing DQ-BSA digestion identified isorhamnetin (3'--methylquercetin) as an active compound. Interestingly, structural comparison to inactive flavonols revealed that the chemical structure of the B-ring moiety in isorhamnetin is the primary determinant of its lysosome-enhancing activity. Unexpectedly isorhamnetin failed to inhibit mTORC1-TFEB signaling, a master regulator of lysosomal biogenesis and function. Our data suggested that the other molecular mechanism might be critical for the regulation of lysosomes in macrophages. ANOVA: analysis of variance; ApoE: apolipoprotein E; ATP6V0D2: ATPase H transporting V0 subunit d2; BAF: bafilomycin A1; BODIPY: boron dipyrromethene; BSA: bovine serum albumin; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco's modified eagle medium; DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LC-MS/MS: liquid chromatography tandem mass spectrometry; MITF: microphthalmia-associated transcription factor; MRM: multiple reaction monitoring; mTORC1: mechanistic target of rapamycin complex 1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor γ; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SDS: sodium dodecyl sulfate; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TBS: Tris-buffered saline; TFA: trifluoroacetic acid; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcriptional factor EB; TFEC: transcription factor EC; V-ATPase: vacuolar-type proton ATPase.

Bioscience, Biotechnology, and Biochemistry


Reduction of stearoyl-CoA desaturase (SCD) contributes muscle atrophy through the excess endoplasmic reticulum stress in chronic kidney disease

Skeletal muscle atrophy is associated with mortality and poor prognosis in patients with chronic kidney disease (CKD). However, underlying mechanism by which CKD causes muscle atrophy has not been completely understood. The quality of lipids (lipoquality), which is defined as the functional features of diverse lipid species, has recently been recognized as the pathology of various diseases. In this study, we investigated the roles of the stearoyl-CoA desaturase (SCD), which catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, in skeletal muscle on muscle atrophy in CKD model animals. In comparison to control rats, CKD rats decreased the SCD activity and its gene expression in atrophic gastrocnemius muscle. Next, oleic acid blocked the reduction of the thickness of C2C12 myotubes and the increase of the endoplasmic reticulum stress induced by SCD inhibitor. Furthermore, endoplasmic reticulum stress inhibitor ameliorated CKD-induced muscle atrophy (the weakness of grip strength and the decrease of muscle fiber size of gastrocnemius muscle) in mice and the reduction of the thickness of C2C12 myotubes by SCD inhibitor. These results suggest that the repression of SCD activity causes muscle atrophy through excessive endoplasmic reticulum stress in CKD.

Journal of Clinical Biochemistry and Nutrition


High-fat diets provoke phosphorus absorption from the small intestine in rats.


Association of increased renal Cyp24a1 gene expression with low plasma 1,25-dihydroxyvitamin D levels in rats with streptozotocin-induced diabetes.

Decreases in plasma vitamin D concentrations have been reported in diabetes, although the mechanism involved in this decrease is unclear. Here, we investigated the association between Cyp24a1, a vitamin D catabolic enzyme, and abnormalities in vitamin D metabolism in streptozotocin-induced diabetes rats, an animal model of type 1 diabetes. Plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] levels were significantly lower in streptozotocin-induced diabetes rats and renal Cyp24a1 mRNA expression levels were increased. Western blotting analysis of streptozotocin-induced diabetes rats kidney tissues with anti-CYP24A1 antibody showed a strong signal around 40 kDa, which differs from the predicted 50-55 kDa molecular weight for full-length Cyp24a1 and could represent the Cyp24a1-splicing variant that lacks exons 1 and 2. We observed high levels of renal Cyp24a1-splicing variant mRNA expression in streptozotocin-induced diabetes rats. We also confirmed transcriptional up-regulation of endogenous Cyp24a1 mRNA expression through glucocorticoid receptors by glucocorticoid in opossum kidney proximal cells. Taken together, our results indicated that high Cyp24a1 expression levels may play a role in the decrease of plasma 1,25(OH)2D levels in streptozotocin-induced diabetes rats. High plasma corticosterone levels in diabetes may affect transcriptional regulation to promote increases in Cyp24a1 expression.


Regulation of α-Klotho Expression by Dietary Phosphate During Growth Periods.

Inorganic phosphate (Pi) is an essential nutrient for maintaining various biological functions, particularly during growth periods. Excess intake of dietary Pi increases the secretion of fibroblast growth factor 23 (FGF23) and parathyroid hormone to maintain plasma Pi levels. FGF23 is a potent phosphaturic factor that binds to the α-klotho/FGFR complex in the kidney to promote excretion of Pi into the urine. In addition, excess intake of dietary Pi decreases renal α-klotho expression. Down-regulation or lack of α-klotho induces a premature aging-like phenotype, resulting from hyperphosphatemia, and leading to conditions such as ectopic calcification and osteoporosis. However, it remains unclear what effects dietary Pi has on α-klotho expression at different life stages, especially during growth periods. To investigate this, we used C57BL/6J mice in two life stages during growing period. Weaned (3 weeks old) and periadolescent (7 weeks old) were randomly divided into seven experimental groups and fed with 0.02, 0.3, 0.6, 0.9, 1.2, 1.5, or 1.8% Pi diets for 7 days. As a result, elevated plasma Pi and FGF23 levels and decreased renal α-klotho expression were observed in weaned mice fed with a high Pi diet. In addition, a high Pi diet clearly induced renal calcification in the weaned mice. However, in the periadolescent group, renal calcification was not observed, even in the 1.8% Pi diet group. The present study indicates that a high Pi diet in weaned mice has much greater adverse effects on renal α-klotho expression and pathogenesis of renal calcification compared with periadolescent mice.


High phosphate diet suppresses lipogenesis in white adipose tissue.

Excessive phosphate intake has been positively associated with renal and vascular dysfunction, conversely negatively associated with body fat accumulation. We investigated the effect of a high-phosphate diet on the expression of lipid metabolic genes in white adipose tissue and liver. Male 8-week-old Sprague-Dawley rats were fed a control diet containing 0.6% phosphate or a high-phosphate diet containing 1.5% phosphate for 4 weeks. In comparison to the control group, the HP group showed a significantly lower body fat mass and fasting plasma insulin level alongside decreased lipogenic and increased lipolytic gene expression in visceral fat. Additionally, the expression of genes involved in hepatic lipogenesis, hepatic glycogenesis, and triglyceride accumulation decreased in the high-phosphate group. Exogenous phosphate, parathyroid hormone, and fibroblast growth factor 23 did not directly affect the expression of lipolytic or lipogenic genes in 3T3-L1 adipocytes and HepG2 hepatocytes. Thus, the high-phosphate diet suppressed the activity of white adipose tissue by increasing lipolytic gene expression and decreasing lipogenic gene expression. These effects could have been caused by the lowered fasting plasma insulin level that occurred in response to the high-phosphate diet, but were not directly caused by the increases in plasma phosphate or phosphaturic hormones.


24-hydroxylase gene expression in renal proximal tubular cells.

) showed dose-dependent reductions in renal Srebp1c and Cyp24a1 mRNA levels. Taken together, our results suggest that SREBP1 trans-activates CYP24A1 expression through SREBP binding elements present in the promoter.

Biochemical and Biophysical Research Communications


Investigation of dose-dependent effects of fat on blood glucose, serum insulin, and appetite sensation.

, respectively. We conducted a randomized, crossover trial and measured laboratory data and appetite sensation via the visual analog scale. Each participant was provided with four different test meals. They consisted of common, basic foods and contained 75 g liquid glucose and 4 slices of crackers to which 0 g butter (control), 10 g butter (B10), 20 g butter (B20), and 40 g butter (B40) were added, respectively. The results indicated that single ingestion of butter did not influence laboratory values of glucose, insulin, glucose-dependent insulinotropic polypeptide (GIP), total bile acids, or high-sensitivity CRP (hs-CRP). Regarding postprandial appetite sensation, appetite ratings for fullness were the highest after the B40 meal (p < 0.05);however, satisfaction ratings were not significantly different after the ingestion of this meal. Ratings were significantly different after the B20 meal. In conclusion, healthy adult subjects experienced fullness and satisfaction after ingesting 20-40 g of butter. J. Med. Invest. 65:203-207, August, 2018.

The Journal of Medical Investigation : JMI


The age-related changes of dietary phosphate responsiveness in plasma 1,25-dihydroxyvitamin D levels and renal Cyp27b1 and Cyp24a1 gene expression is associated with renal -Klotho gene expression in mice.

mRNA expression showed a significant negative correlation with plasma 1,25-dihydroxyvitamin D levels in the high phosphate group. Our results suggest that age-related alterations in renal -Klotho expression could affect the responsiveness of dietary phosphate to vitamin D metabolism.


Dietary phosphate exacerbates intestinal inflammation in experimental colitis.

study, the effects of phosphate on proinflammatory cytokine induction and reactive oxygen species production in RAW264.7 macrophage were examined. Dietary phosphate exacerbated intestinal inflammation in experimental colitis in a dose-dependent manner, as assessed by the clinical disease activity score, colon length, and histology. Furthermore, the high phosphate diet increased myeloperoxidase activity and proinflammatory cytokine mRNA expression through the activation of nuclear factor B in the inflamed colon. In addition, high phosphate loading in RAW264.7 cells directly enhanced reactive oxygen species production and proinflammatory cytokine gene expression. Our results demonstrated that the high phosphate diet exacerbated intestinal inflammation in experimental colitis. These findings have important therapeutic implications for inflammatory bowel disease patients.

Journal of Clinical Biochemistry and Nutrition


Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells.

Emerging evidence indicates that upregulation of the ER stress-induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell-specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBP . These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs.


Effects of dietary phosphate on glucose and lipid metabolism.

Recent epidemiological and animal studies have suggested that excess intake of phosphate (Pi) is a risk factor for the progression of chronic kidney disease and its cardiovascular complications. However, little is known about the impact of dietary high Pi intake on the development of metabolic disorders such as obesity and type II diabetes. In this study, we investigated the effects of dietary Pi on glucose and lipid metabolism in healthy rats. Male, 8-wk-old Sprague-Dawley rats were divided into 3 groups and given experimental diets containing varying amounts of Pi, i.e. 0.2% (low Pi, LP), 0.6% (control Pi, CP), and 1.2% (high Pi, HP). After 4 weeks, HP group showed lower visceral fat accumulation compared with other groups, accompanied by a low respiratory exchange ratio (VCO2/VO2) without alteration of locomotive activity. HP group had lower levels of plasma insulin and non-esterified fatty acids. In addition, HP group also showed suppressed expression of hepatic lipogenic genes including sterol regulatory element binding protein-1c, fatty acid synthase, and acetyl-CoA carboxylase, whereas there was no difference in hepatic fat oxidation among the groups. On the other hand, uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator alpha (PGC1) expression were significantly increased in the brown adipose tissue (BAT) of HP group. Our data demonstrated that a high Pi diet can negatively regulate lipid synthesis in the liver, and increased mRNA expression related to lipid oxidation and UCP1 in BAT, thereby preventing visceral fat accumulation. Thus, dietary Pi is a novel metabolic regulator.

American Journal of Physiology, Endocrinology and Metabolism


Saturated phosphatidic acids mediate saturated fatty acid-induced vascular calcification and lipotoxicity.

Recent evidence indicates that saturated fatty acid-induced (SFA-induced) lipotoxicity contributes to the pathogenesis of cardiovascular and metabolic diseases; however, the molecular mechanisms that underlie SFA-induced lipotoxicity remain unclear. Here, we have shown that repression of stearoyl-CoA desaturase (SCD) enzymes, which regulate the intracellular balance of SFAs and unsaturated FAs, and the subsequent accumulation of SFAs in vascular smooth muscle cells (VSMCs), are characteristic events in the development of vascular calcification. We evaluated whether SMC-specific inhibition of SCD and the resulting SFA accumulation plays a causative role in the pathogenesis of vascular calcification and generated mice with SMC-specific deletion of both Scd1 and Scd2. Mice lacking both SCD1 and SCD2 in SMCs displayed severe vascular calcification with increased ER stress. Moreover, we employed shRNA library screening and radiolabeling approaches, as well as in vitro and in vivo lipidomic analysis, and determined that fully saturated phosphatidic acids such as 1,2-distearoyl-PA (18:0/18:0-PA) mediate SFA-induced lipotoxicity and vascular calcification. Together, these results identify a key lipogenic pathway in SMCs that mediates vascular calcification.


Dietary phosphate supplementation delays the onset of iron deficiency anemia and affects iron status in rats.

Inorganic phosphate (Pi) plays critical roles in bone metabolism and is an essential component of 2,3-diphosphoglycerate (2,3-DPG). It has been reported that animals fed a low-iron diet modulate Pi metabolism, whereas the effect of dietary Pi on iron metabolism, particularly in iron deficiency anemia (IDA), is not fully understood. In this study, we hypothesized the presence of a link between Pi and iron metabolism and tested the hypothesis by investigating the effects of dietary Pi on iron status and IDA. Wistar rats aged 4 weeks were randomly assigned to 1 of 4 experimental dietary groups: normal iron content (Con Fe)+0.5% Pi, low-iron (Low Fe)+0.5% Pi, Con Fe+1.5% Pi, and Low Fe+1.5% Pi. Rats fed the 1.5% Pi diet for 14 days, but not for 28 days, maintained their anemia state and plasma erythropoietin concentrations within the reference range, even under conditions of low iron. In addition, plasma concentrations of 2,3-DPG were significantly increased by the 1.5% Pi diets and were positively correlated with plasma Pi concentration (r=0.779; P<.001). Dietary Pi regulated the messenger RNA expression of iron-regulated genes, including divalent metal transporter 1, duodenal cytochrome B, and hepcidin. Furthermore, iron concentration in liver tissues was increased by the 1.5% Pi in Con Fe diet. These results suggest that dietary Pi supplementation delays the onset of IDA and increases plasma 2,3-DPG concentration, followed by modulation of the expression of iron-regulated genes.


Chronic kidney disease (CKD) is an increasing problem worldwide. The number of end-stage renal disease patients requiring treatment by dialysis is estimated to be increasing by 10,000 patients per year in Japan. Furthermore, an estimated 13 million people are living with CKD in Japan. Various complications are associated with CKD, including cardiovascular disease (CVD). More than one-third of CKD patients die from CVD. Thus, prevention of CVD is a primary concern for the treatment of CKD patients. CKD-mineral and bone disorder (CKD-MBD) is a serious complication that typically leads to CVD. Hyperphosphatemia is thought to be a central-risk factor for CKD-MBD. Therefore, managing hyperphosphatemia is crucial to prevent CKD-MBD and CVD. It is difficult to achieve the target serum phosphate level through dietary modifications alone in patients with hyperphosphatemia, because most foods contain phosphate. Thus, phosphate binders such as calcium carbonate are commonly prescribed to CKD patients with hyperphosphatemia, but these have undesirable side effects. Inhibition of intestinal phosphate transport activity has also been investigated as an alternative approach for controlling serum phosphate levels in CKD patients. Nicotinamide, which is the amide of niacin, can inhibit intestinal phosphate transport. Niacin and related compounds have also been developed as drugs for hyperlipidemia conditions, especially hypertriglyceridemia with low high-density lipoprotein. This type of dyslipidemia is frequently observed in CKD patients and is a modifiable risk factor for CVD. Thus, niacin and related compounds may have utility for the treatment of both hyperphosphatemia and dyslipidemia in CKD patients to prevent CVD.

Journal of Nutritional Science and Vitaminology


Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE-/- mice and LDLR-/- mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE-/- and LDLR-/- mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE-/- mice through the inactivation of NF-B. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-B, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.


Endoplasmic reticulum stress effector CCAAT/enhancer-binding protein homologous protein (CHOP) regulates chronic kidney disease-induced vascular calcification.

Cardiovascular diseases such as atherosclerosis and vascular calcification are a major cause of death in patients with chronic kidney disease (CKD). Recently, the long-awaited results of the Study of Heart and Renal Protection trial were reported. This large randomized clinical trial found that an extensive cholesterol-lowering therapy through the combination of simvastatin and ezetimibe significantly reduced cardiovascular diseases in a wide range of patients with CKD. However, the mechanism by which this cholesterol-lowering therapy reduces CKD-dependent vascular diseases remains elusive. The objective of the present study was to determine the contribution of the oxysterol-induced pro-apoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) on the pathogenesis of CKD-dependent cardiovascular diseases through endoplasmic reticulum stress signaling. CKD increased levels of serum oxysterols such as 7-ketocholesterol in human patients and ApoE(-/-) mice. Treatment with simvastatin plus ezetimibe strongly reduced levels of serum oxysterols and attenuated CKD-dependent atherosclerosis, vascular cell death, vascular calcification, and cardiac dysfunction. This therapy also reduced aortic endoplasmic reticulum stress induced by CKD. The short hairpin RNA-mediated knockdown of CHOP and activating transcription factor-4 in vascular smooth muscle cells attenuated oxysterol-induced mineralization, osteogenic differentiation, and endoplasmic reticulum stress. In addition, CHOP deficiency protected ApoE(-/-) mice from CKD-dependent vascular calcification, cardiac dysfunction, and vascular cell death. These data reveal that the cholesterol-lowering therapy of simvastatin plus ezetimibe attenuates CKD-dependent vascular diseases through a reduction of oxysterol-mediated endoplasmic reticulum stress. CHOP plays a crucial role in the pathogenesis of CKD-dependent vascular calcification.

Journal of the American Heart Association


Short-term dietary phosphate restriction up-regulates ileal fibroblast growth factor 15 gene expression in mice.

Members of the fibroblast growth factor (FGF) 19 subfamily, including FGF23, FGF15/19, and FGF21, have a role as endocrine factors which influence the metabolism of inorganic phosphate (Pi) and vitamin D, bile acid, and energy. It has been reported that dietary Pi regulates circulating FGF23. In this study, the short-term effects of dietary Pi restriction on the expression of FGF19 subfamily members in mice were analyzed. An initial analysis confirmed plasma FGF23 levels positively correlated with the amount of dietary Pi. On the other hand, ileal Fgf15 gene expression, but not hepatic Fgf21 gene expression, was up-regulated by dietary Pi restriction. In addition, we observed the increase of plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] levels by dietary Pi restriction, and the up-regulation of ileal Fgf15 mRNA expression by 1,25(OH)2D3 and vitamin D receptor (VDR). Importantly, dietary Pi restriction-induced Fgf15 gene expression was prevented in VDR-knockout mice. Furthermore, diurnal variations of plasma triglyceride concentrations and hepatic mRNA expression of the bile acid synthesis enzyme Cyp7a1 as one of Fgf15 negative target genes was influenced by dietary Pi restriction. These results suggest that dietary Pi restriction up-regulates ileal Fgf15 gene expression through 1,25(OH)2D3 and VDR, and may affect hepatic bile acid homeostasis.

Journal of Clinical Biochemistry and Nutrition


Downregulation of renal type IIa sodium-dependent phosphate cotransporter during lipopolysaccharide-induced acute inflammation.

The type IIa sodium-dependent phosphate cotransporter (Npt2a) plays a critical role in reabsorption of inorganic phosphate (Pi) by renal proximal tubular cells. Pi abnormalities during early stages of sepsis have been reported, but the mechanisms regulating Pi homeostasis during acute inflammation are poorly understood. We examined the regulation of Pi metabolism and renal Npt2a expression during lipopolysaccharide (LPS)-induced inflammation in mice. Dose-response and time-course studies with LPS showed significant increases of plasma Pi and intact parathyroid hormone (iPTH) levels and renal Pi excretion, while renal calcium excretion was significantly decreased. There was no difference in plasma 1,25-dihydroxyvitamin D levels, but the induction of plasma intact fibroblast growth factor 23 levels peaked 3 h after LPS treatment. Western blotting, immunostaining, and quantitative real-time PCR showed that LPS administration significantly decreased Npt2a protein expression in the brush border membrane (BBM) 3 h after injection, but there was no change in renal Npt2a mRNA levels. Moreover, tumor necrosis factor- injection also increased plasma iPTH and decreased renal BBM Npt2a expression. Importantly, we revealed that parathyroidectomized rats had impaired renal Pi excretion and BBM Npt2a expression in response to LPS. These results suggest that the downregulation of Npt2a expression in renal BBM through induction of plasma iPTH levels alter Pi homeostasis during LPS-induced acute inflammation.

American Journal of Physiology, Renal Physiology


PERK-eIF2-ATF4-CHOP signaling contributes to TNF-induced vascular calcification.

Vascular calcification is a common feature in patients with chronic kidney disease (CKD). CKD increases serum levels of tumor necrosis factor- (TNF), a critical mediator of vascular calcification. However, the molecular mechanism by which TNF promotes CKD-dependent vascular calcification remains obscure. The purpose of the present study was to investigate whether TNF-induced vascular calcification in CKD is caused by the endoplasmic reticulum response involving protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2 (eIF2), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). We examined the effects of TNF on the endoplasmic reticulum (ER) stress response of vascular smooth muscle cells (VSMCs). TNF treatment drastically induced the PERK-eIF2-ATF4-CHOP axis of the ER stress response in VSMCs. PERK, ATF4, and CHOP shRNA-mediated knockdowns drastically inhibited mineralization and osteogenesis of VSMCs induced by TNF. CKD induced by 5/6 nephrectomies activated the PERK-eIF2-ATF4-CHOP axis of the ER stress response in the aortas of ApoE-/- mice with increased aortic TNF expression and vascular calcification. Treatment of 5/6 nephrectomized ApoE-/- mice with the TNF neutralizing antibody or chemical Chaperones reduced aortic PERK-eIF2-ATF4-CHOP signaling of the ER stress increased by CKD. This resulted in the inhibition of CKD-dependent vascular calcification. These results suggest that TNF induces the PERK-eIF2-ATF4-CHOP axis of the ER stress response, leading to CKD-dependent vascular calcification.


Hypercholesterolemia and effects of high cholesterol diet in type IIa sodium-dependent phosphate co-transporter (Npt2a) deficient mice.

The type IIa sodium-dependent phosphate co-transporter (Npt2a) is important to maintain renal inorganic phosphate (Pi) homeostasis and the plasma Pi levels. It has reported that disorder of Pi metabolism in kidney can be risk factors for cardiovascular disease as well as hypercholesterolemia. However, the relationship between Pi and cholesterol metabolism has not been clarified. The current study investigated the effects of Npt2a gene ablation that is known as hypophosphatemia model on cholesterol metabolism in mice. Npt2a deficient (Npt2a(-/-)) mice and wild type mice were fed diets with or without 2% cholesterol for 12 days. Plasma lipid and lipoprotein profile analysis revealed that plasma lipid levels (total, LDL and HDL cholesterol) were significantly higher in Npt2a(-/-) mice than wild type (WT) mice. Interestingly, high cholesterol diet markedly increased plasma levels of total, LDL and HDL cholesterol in WT mice, but not Npt2a(-/-) mice. On the other hand, there were no differences in body and liver weight, intake and hepatic lipid accumulation between WT and Npt2a(-/-) mice. These results suggest that ablation of Npt2a gene induces hypercholesterolemia and affects the ability to respond normally to dietary cholesterol.

The Journal of Medical Investigation : JMI


Activating transcription factor 4 regulates stearate-induced vascular calcification.

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2 pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate.


Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.

Vascular calcification is recognized as an independent predictor of cardiovascular mortality, particularly in subjects with chronic kidney disease. However, the pathways by which dysregulation of lipid and mineral metabolism simultaneously occur in this particular population remain unclear. We have shown that activation of the farnesoid X receptor (FXR) blocks mineralization of bovine calcifying vascular cells (CVCs) and in ApoE knock-out mice with 5/6 nephrectomy. In contrast to FXR, this study showed that liver X receptor (LXR) activation by LXR agonists and adenovirus-mediated LXR overexpression by VP16-LXR and VP16-LXR accelerated mineralization of CVCs. Conversely, LXR inhibition by dominant negative (DN) forms of LXR and LXR reduced calcium content in CVCs. The regulation of mineralization by FXR and LXR agonists was highly correlated with changes in lipid accumulation, fatty acid synthesis, and the expression of sterol regulatory element binding protein-1 (SREBP-1). The rate of lipogenesis in CVCs through the SREBP-1c dependent pathway was reduced by FXR activation, but increased by LXR activation. SREBP-1c overexpression augmented mineralization in CVCs, whereas SREBP-1c DN inhibited alkaline phosphatase activity and mineralization induced by LXR agonists. LXR and SREBP-1c activations increased, whereas FXR activation decreased, saturated and monounsaturated fatty acids derived from lipogenesis. In addition, we found that stearate markedly promoted mineralization of CVCs as compared with other fatty acids. Furthermore, inhibition of either acetyl-CoA carboxylase or acyl-CoA synthetase reduced mineralization of CVCs, whereas inhibition of stearoyl-CoA desaturase induced mineralization. Therefore, a stearate metabolite derived from lipogenesis might be a risk factor for the development of vascular calcification.


最前線 慢性腎臓病の合併症とlipotoxicity

最前線 慢性腎臓病の合併症とlipotoxicity