Yuji Kamijo

Constitutive Regulation of Cardiac Fatty Acid Metabolism through Peroxisome Proliferator-activated Receptor α Associated with Age-dependent Cardiac Toxicity

The peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear receptor superfamily and mediates the biological effects of peroxisome proliferators. To determine the physiological role of PPARα in cardiac fatty acid metabolism, we examined the regulation of expression of cardiac fatty acid-metabolizing proteins using PPARα-null mice. The capacity for constitutive myocardial β-oxidation of the medium and long chain fatty acids, octanoic acid and palmitic acid, was markedly reduced in the PPARα-null mice as compared with the wild-type mice, indicating that mitochondrial fatty acid catabolism is impaired in the absence of PPARα. In contrast, constitutive β-oxidation of the very long chain fatty acid, lignoceric acid, did not differ between the mice, suggesting that the constitutive expression of enzymes involved in peroxisomal β-oxidation is independent of PPARα.Indeed, PPARα-null mice had normal levels of the peroxisomal β-oxidation enzymes except the D-type bifunctional protein. At least seven mitochondrial fatty acid-metabolizing enzymes were expressed at much lower levels in the PPARα-null mice, whereas other fatty acid-metabolizing enzymes were present at similar or slightly lower levels in the PPARα-null, as compared with wild-type mice. Additionally, lower constitutive mRNA expression levels of fatty acid transporters were found in the PPARα-null mice, suggesting a role for PPARα in fatty acid transport and catabolism. Indeed, in fatty acid metabolism experiments in vivo, myocardial uptake of iodophenyl 9-methylpentadecanoic acid and its conversion to 3-methylnonanoic acid were reduced in the PPARα-null mice. Interestingly, a decreased ATP concentration after exposure to stress, abnormal cristae of the mitochondria, abnormal caveolae, and fibrosis were observed only in the myocardium of the PPARα-null mice. These cardiac abnormalities appeared to proceed in an age-dependent manner. Taken together, the results presented here indicate that PPARα controls constitutive fatty acid oxidation, thus establishing a role for the receptor in cardiac fatty acid homeostasis. Furthermore, altered expression of fatty acid-metabolizing proteins seems to lead to myocardial damage and fibrosis, as inflammation and abnormal cell growth control can cause these conditions.

Identification of Functions of Peroxisome Proliferator-Activated Receptor α in Proximal Tubules

Peroxisome proliferator-activated receptor alpha(PPARalpha) is a member of the steroid/nuclear receptor superfamily that is intensively expressed in the kidney, but its physiologic function is unknown. In this study, PPARalpha-null mice were used to help clarify the function. Starved PPARalpha-null mice were found to secrete significantly more quantities of urine albumin than starved wild-type mice. Furthermore, the appearance of giant lysosomes, marked accumulation of albumin, and an impaired ability concerning albumin digestion were found only in proximal tubules of the starved PPARalpha-null mice. These abnormalities were probably derived from ATP insufficiency as a result of the starvation-induced decline of carbohydrate metabolism and a lack of PPARalpha-dependent fatty acid metabolism. It is interesting that these abnormalities disappeared when glucose was administered. Taken together, these findings demonstrate important functions of PPARalpha in the proximal tubules, the dynamic regulation of the protein-degradation system through maintenance of ATP homeostasis, and emphasize the importance of the fatty acid metabolism in renal physiology.

Eicosapentaenoic acid lowers plasma and liver cholesterol levels in the presence of peroxisome proliferators-activated receptor alpha

Eicosapentaenoic acid (EPA) is known to lower plasma cholesterol level and triglycerides, but its precise molecular mechanisms have not been reported. The objective of this study was to determine the mechanism of action of EPA in lowering plasma cholesterol and triglyceride levels. In this study, we found that long-term, highly purified EPA administration effectively reduced plasma and hepatic cholesterol levels in wild-type mice but not in peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice. The significant down-regulation was detected at the transcriptional level on genes involved in cholesterol biosynthesis and cholesterol efflux in the liver only in wild-type mice. Limited changes were found in molecules involved in lipoprotein assembly and uptake, intracellular cholesterol transport, bile acid biosynthesis, and bile secretion. Transcription factors regulating cholesterol homeostasis were insignificantly modulated by the EPA treatment, except for sterol response element-binding protein-2 (SREBP-2). Based on these findings, EPA potentially lowers the plasma cholesterol levels by suppressing gene expression of cholesterol biosynthesis enzymes and a cholesterol efflux protein from the liver. In mature SREBP-2, processing ability appears to play an important role in the presence of PPARalpha. Our study provides novel evidence of an additional rationale for the use of EPA in the prevention and treatment of hypercholesterolemia.

PPAR Protects Proximal Tubular Cells from Acute Fatty Acid Toxicity

Fatty acids bound to albumin are filtered through glomeruli, reabsorbed by proximal tubular epithelial cells, and metabolized. Because albumin serves as a carrier, an increase in delivery of fatty acids to the proximal tubule may occur in proteinuric states, possibly leading to toxic effects. At

Bezafibrate at Clinically Relevant Doses Decreases Serum/Liver Triglycerides via Down-Regulation of Sterol Regulatory Element-Binding Protein-1c in Mice: A Novel Peroxisome Proliferator-Activated Receptor α-Independent Mechanism

The triglyceride-lowering effect of bezafibrate in humans has been attributed to peroxisome proliferator-activated receptor (PPAR) α activation based on results from rodent studies. However, the bezafibrate dosages used in conventional rodent experiments are typically higher than those in clinical use (≥50 versus ≤10 mg/kg/day), and thus it remains unclear whether such data can be translated to humans. Furthermore, because bezafibrate is a pan-PPAR activator, the actual contribution of PPARα to its triglyceride-lowering properties remains undetermined. To address these issues, bezafibrate at clinically relevant doses (10 mg/kg/day; low) was administered to wild-type and Ppara-null mice, and its effects were compared with those from conventionally used doses (100 mg/kg/day; high). Pharmacokinetic analyses showed that maximum plasma concentration and area under the concentration-time curve in bezafibrate-treated mice were similar to those in humans at low doses, but not at high doses. Low-dose bezafibrate decreased serum/liver triglycerides in a PPARα-independent manner by attenuation of hepatic lipogenesis and triglyceride secretion. It is noteworthy that instead of PPAR activation, down-regulation of sterol regulatory element-binding protein (SREBP)-1c was observed in mice undergoing low-dose treatment. High-dose bezafibrate decreased serum/liver triglycerides by enhancement of hepatic fatty acid uptake and β-oxidation via PPARα activation, as expected. In conclusion, clinically relevant doses of bezafibrate exert a triglyceride-lowering effect by suppression of the SREBP-1c-regulated pathway in mice and not by PPARα activation. Our results may provide novel information about the pharmacological mechanism of bezafibrate action and new insights into the treatment of disorders involving SREBP-1c.

PPARα attenuates the proinflammatory response in activated mesangial cells

The activated mesangial cell is an important therapeutic target for the control of glomerulonephritis. The peroxisome proliferator-activated receptor alpha (PPARalpha) has attracted considerable attention for its anti-inflammatory effects; however, its roles in the mesangial cells remain unknown. To determine the anti-inflammatory function of PPARalpha in mesangial cells, wild-type and Ppara-null cultured mesangial cells were exposed to lipopolysaccharide (LPS). LPS treatment caused enhanced proinflammatory responses in the Ppara-null cells compared with wild-type cells, as revealed by the induction of interleukin-6, enhanced cell proliferation, and the activation of the nuclear factor (NF)-kappaB signaling pathway. In wild-type cells resistant to inflammation, constitutive expression of PPARalpha was undetectable. However, LPS treatment induced the significant appearance and substantial activation of PPARalpha, which would attenuate the proinflammatory responses through its antagonizing effects on the NF-kappaB signaling pathway. The induction of PPARalpha was coincident with the appearance of alpha-smooth muscle actin, which might be associated with the phenotypic changes of mesangial cells. Moreover, another examination using LPS-injected wild-type mice demonstrated the appearance of PPARalpha-positive cells in glomeruli, suggesting in vivo correlation with PPARalpha induction. These results suggest that PPARalpha plays crucial roles in the attenuation of inflammatory response in activated mesangial cells. PPARalpha might be a novel therapeutic target against glomerular diseases.

Significance of chemokine receptor expression in aggressive NK cell leukemia

Natural killer (NK) cell-type lymphoproliferative diseases of granular lymphocytes can be subdivided into aggressive NK cell leukemia (ANKL) and chronic NK cell lymphocytosis (CNKL). One reason for the poor outcome in ANKL is leukemic infiltration into multiple organs. The mechanisms of cell trafficking associated with the chemokine system have been investigated in NK cells. To clarify the mechanism of systemic migration of leukemic NK cells, we enrolled nine ANKL and six CNKL cases, and analyzed the expression profiles and functions of chemokine receptors by flowcytometry and chemotaxis assay. CXCR1 was detected on NK cells in all groups, and CCR5 was positive in all ANKL cells. Proliferating NK cells were simultaneously positive for CXCR1 and CCR5 in all ANKL patients examined, and NK cells with this phenotype did not expand in CNKL patients or healthy donors. ANKL cells showed enhanced chemotaxis toward the ligands of these receptors. These results indicated that the chemokine system might play an important role in the pathophysiology of ANKL and that chemokine receptor profiling might be a novel tool for discriminating ANKL cells from benign NK cells.

Peroxisome Proliferator-Activated Receptor Protects against Glomerulonephritis Induced by Long-Term Exposure to the Plasticizer Di-(2-Ethylhexyl)Phthalate

Safety concerns about di-(2-ethylhexyl)phthalate (DEHP), a plasticizer and a probable endocrine disruptor, have attracted considerable public attention, but there are few studies about long-term exposure to DEHP. DEHP toxicity is thought to involve peroxisome proliferator–activated receptor α (PPARα), but this contention remains controversial. For investigation of the long-term toxicity of DEHP and determination of whether PPARα mediates toxicity, wild-type and PPARα-null mice were fed a diet that contained 0.05 or 0.01% DEHP for 22 mo. PPARα-null mice that were exposed to DEHP exhibited prominent immune complex glomerulonephritis, most likely related to elevated glomerular oxidative stress. Elevated NADPH oxidase, low antioxidant enzymes, and absence of the PPARα-dependent anti-inflammatory effects that normally antagonize the NFκB signaling pathway accompanied the glomerulonephritis in PPARα-null mice. The results reported here indicate that PPARα protects against the nephrotoxic effects of long-term exposure to DEHP.

Acute kidney injury induced by protein-overload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides

Sulfatides, possible antithrombotic factors belonging to sphingoglycolipids, are widely distributed in mammalian tissues and serum. We recently found that the level of serum sulfatides was significantly lower in hemodialysis patients than that in normal subjects, and that the serum level closely correlated to the incidence of cardiovascular disease. These findings suggest a relationship between the level of serum sulfatides and kidney function; however, the molecular mechanism underlying this relationship remains unclear. In the present study, the influence of kidney dysfunction on the metabolism of sulfatides was examined using an established murine model of acute kidney injury, protein-overload nephropathy in mice. Protein-overload treatment caused severe proximal tubular injuries within 4days, and this treatment obviously decreased both serum and hepatic sulfatide levels. The sphingoid composition of serum sulfatides was very similar to that of hepatic ones at each time point, suggesting that the serum sulfatide level is dependent on the hepatic secretory ability of sulfatides. The treatment also decreased hepatic expression of cerebroside sulfotransferase (CST), a key enzyme in sulfatide metabolism, while it scarcely influenced the expression of the other sulfatide-metabolizing enzymes, including arylsulfatase A, ceramide galactosyltransferase, and galactosylceramidase. Pro-inflammatory responses were not detected in the liver of these mice; however, potential oxidative stress was increased. These results suggest that down-regulation of hepatic CST expression, probably affected by oxidative stress from kidney injury, causes reduction in liver and serum sulfatide levels. This novel mechanism, indicating the crosstalk between kidney injury and specific liver function, may prove useful for helping to understand the situation where human hemodialysis patients have low levels of serum sulfatides.

Cholesterol-lowering effect of bezafibrate is independent of peroxisome proliferator-activated receptor activation in mice

The hypocholesterolemic potential of peroxisome proliferator-activated receptor (PPAR) pan-activator bezafibrate has been documented. However, in addition to uncertainty about the contribution of PPAR alpha to its effect, there is a marked discrepancy in bezafibrate dosages used in previous rodent experiments (> or = 50 mg/kg/day) and those in clinical use (< or = 10 mg/kg/day). To investigate the association between bezafibrate-induced cholesterol reduction and PPAR alpha activation, wild-type and Ppar a-null mice were treated with bezafibrate at high (100 mg/kg/day) or low (10 mg/kg/day) doses and analyzed. High-dose treatment decreased hepatic cholesterol content in wild-type mice, but increased serum cholesterol concentration. In liver samples, simultaneous increases in the expression of numerous proteins involved in cholesterol biosynthesis and catabolism, as well as cholesterol influx and efflux, were observed, which made interpretation of phenotype changes subtle. These complicated responses were believed to be associated with intensive PPAR activation and accompanying up-regulation of liver X receptor alpha, farnesoid X receptor, and sterol regulatory element-binding protein 2 (SREBP2). In contrast, low-dose bezafibrate treatment decreased serum and hepatic cholesterol concentrations in a PPAR alpha-independent manner, probably from suppression of SREBP2-regulated cholesterogenesis and enhancement of cholesterol catabolism due to elevated 7alpha-hydroxylase levels. Interestingly, the low-dose treatment did not affect the expression of PPAR target genes or number of peroxisomes, suggesting the absence of PPAR activation. These results demonstrate that the action of bezafibrate on cholesterol metabolism may vary with dosage, and that the cholesterol-reducing effect found in mice at dosages similar to those administered to humans is independent of significant PPAR activation.