In this study, we defined the role of peroxisome proliferator-activated receptor ␤͞␦ (PPAR␦) in metabolic homeostasis by using subtype selective agonists. Analysis of rat L6 myotubes treated with the PPAR␦ subtype-selective agonist, GW501516, by the Affymetrix oligonucleotide microarrays revealed that PPAR␦ controls fatty acid oxidation by regulating genes involved in fatty acid transport, ␤-oxidation, and mitochondrial respiration. Similar PPAR␦-mediated gene activation was observed in the skeletal muscle of GW501516-treated mice. Accordingly, GW501516 treatment induced fatty acid ␤-oxidation in L6 myotubes as well as in mouse skeletal muscles. Administration of GW501516 to mice fed a high-fat diet ameliorated diet-induced obesity and insulin resistance, an effect accompanied by enhanced metabolic rate and fatty acid ␤-oxidation, proliferation of mitochondria, and a marked reduction of lipid droplets in skeletal muscles. Despite a modest body weight change relative to vehicle-treated mice, GW501516 treatment also markedly improved diabetes as revealed by the decrease in plasma glucose and blood insulin levels in genetically obese ob͞ob mice. These data suggest that PPAR␦ is pivotal to control the program for fatty acid oxidation in the skeletal muscle, thereby ameliorating obesity and insulin resistance through its activation in obese animals.obesity ͉ insulin resistance ͉ thermogenesis ͉ pancreatic ␤-cell ͉ PGC-1␣

A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca 2؉ in response to glucose, and thereby glucoseinduced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5؊͞؊ islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5؊͞؊ islets. Furthermore, exposure of LRP5؉͞؉ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that Wnt͞LRP5 signaling contributes to the glucose-induced insulin secretion in the islets.and LRP6 are coreceptors involved in the Wnt signaling pathway (1-6). The Wnt signaling pathway plays a pivotal role in embryonic development (7,8) and oncogenesis (9) through various signaling molecules including Frizzled receptors (10), recently characterized LRP5 and LRP6 (1-6), and Dickkopf proteins (4, 6). In addition, the Wnt signaling is also involved in adipogenesis by negatively regulating adipogenic transcription factors (Tcfs) (11). Although Wnt signaling has been characterized in both developmental and oncogenic processes, little is known about its function in the normal adult.Recent studies have revealed that loss of function mutations in the LRP5 gene cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (12). LRP5 is expressed in osteoblasts and transduces Wnt signaling via the canonical pathway, thereby modulating bone accrual development (12, 13). A point mutation in a ''propeller'' motif in LRP5 causes a dominant-positive high bone density by impairing the action of a normal antagonist of the Wnt pathway, Dickkopf, thereby increasing Wnt signaling (14,15). In addition, the human LRP5 gene is mapped within the region (IDDM4) linked to type 1 diabetes on chromosome 11q13 (16).In previous studies, we and others showed that LRP5 is highly expressed in many tissues, including hepatocytes and pancreatic beta cells (17,18). We also showed that LRP5 can bind apolipoprotein E (apoE) (18). This finding raises the possibility that LRP5 plays a role in the hepatic clearance of apoE-containing chylomicron remnants, a major plasma lipoprotein carrying diet-derived cholesterol.To evaluate the in vivo roles of LRP5, we generated LRP...

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain-to-blood transport of indoxyl sulfate at the blood-brain barrier (BBB) using the Brain Efflux Index method. [ 3 H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 · 10 )2 /min, and the process is saturable with a K m of 298 lM. This process is inhibited by para-aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acid and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. RT-PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [ 3 H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3-methoxy-4-hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6-mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain-to-blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brainto-blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain. Keywords: blood-brain barrier, efflux transport, indoxyl sulfate, organic anion transporter 3 (OAT3), uremic toxin.

Abstract:We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) 3 H]DHEAS across the BBB (118 l/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 l/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [3 H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K m of 34.4 M and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [ 3 H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [ 3 H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB. Key Words: Blood-brain barrier-Dehydroepiandrosterone sulfate -Efflux transport-Organic anion transporting polypeptide 2-Conditionally immortalized mouse brain capillary endothelial cell line.

These results suggest that rOAT3 is responsible for the renal uptake of indoxyl sulfate, and uremic toxins share the transport mechanism for indoxyl sulfate. Mutual inhibition of these uremic toxins via OAT3 may accelerate their accumulation in the body and, thereby, the progression of nephrotoxicity in uremia.

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had ϳ60% higher plasma cholesterol levels compared with the agematched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE; LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were ϳ3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced arthrosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.Genetic defects in the catabolism of plasma lipoproteins are important causes of hypercholesterolemia and atherosclerosis in humans. The prototypic diseases are familial hypercholesterolemia, caused by a defect in the LDL 1 receptor (LDLR) (1), and familial type III hyperlipoproteinemia, caused by a defect in one of the ligands for LDLR, apolipoprotein E (apoE) (2).ApoE is hypothesized to mediate lipoprotein clearance by binding two receptors: (i) LDLR and (ii) a hepatic chylomicron remnant receptor. ApoE-deficient mice (3-5) and LDLR-deficient mice (6) exhibit hypercholesterolemia, but the severity and manifestations differ markedly. On a normal laboratory chow diet, the apoE knockout mice have much more profound hypercholesterolemia and develop spontaneous atherosclerosis (4).LDL receptor-related protein 5 (LRP5) is a member of the LDL receptor family that are characterized by the presence of cysteine-rich complement type ligand binding domains. LRP5 binds apoE-containing lipoproteins in vitro and is widely expressed in many tissues including hepatocytes, adrenal gland, and pancreas (7).LRP5 and its homologue, LRP6, are postulated to play as co-receptors for Wnt receptors, Frizzled (8 -13). The Wnt signaling pathway plays an essential role in embryonic development (14, 15) and oncogenesis (16) through various signaling molecules including Frizzled receptors (17), , and Dickkopf protei...

The brain efflux index method has been used to clarify the mechanism of efflux transport of acidic amino acids such as L-aspartic acid (L-Asp), L-

Acetyl-CoA synthetase 2 (AceCS2) produces acetylCoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5 -flanking region of the mouse AceCS2 gene demonstrates that Krü ppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.Acetyl-CoA is an important intermediate in various metabolic pathways including fatty acid and cholesterol biosynthesis and the energy production by the citric acid cycle. There are several enzymes that generate acetyl-CoA in the mammals, including pyruvate dehydrogenase, which converts pyruvate to acetyl-CoA without generating free acetate. The degradation of fatty acid via ␤-oxidation system also produces acetyl-CoA as an end product. Although acetate is not an essential source of acetyl-CoA in animals, the enzymatic ligation for the production of acetyl-CoA from acetate and CoA is a key reaction in the catabolism of acetate formed by several conditions including bacterial fermentation in the colon, oxidation of ingested ethanol in the liver. Acetyl-CoA synthetase (AceCS, 1 EC 6.2.1.1) is an enzyme that catalyzes the production of acetyl-CoA from acetate and CoA. In particular, this enzyme plays a key role in the nervous system for recycling of acetate released by acetylcholine esterase for the formation and release of acetylcholine in cholinergic nerve terminals.There are two AceCSs with similar enzymatic properties in mammals: one designated AceCS1 is a cytosolic enzyme and the other, designated AceCS2, is a mitochondrial matrix enzyme (1). Localized in the cytoplasm, AceCS1 provides acetylCoA for the synthesis of fatty acids and cholesterol. In contrast, AceCS2 produces acetyl-CoA for oxida...