Takero Nakajima

Down-regulation of SREBP-1c is associated with the development of burned-out NASH.

BACKGROUND & AIMS It is well-known that hepatic triglycerides (TG) diminish with the progression of non-alcoholic steatohepatitis (NASH), which has been designated as burned-out NASH, but its mechanism remains unclear. We aimed to explore the changes in hepatic fatty acid (FA) and TG metabolism with disease progression. METHODS Hepatic expression of key genes in healthy individuals (n=6) and patients with simple steatosis (SS, n=10), mild NASH (fibrosis stage 1-2, n=20), and advanced NASH (fibrosis stage 3-4, n=20) were assessed by quantitative polymerase chain reaction. RESULTS Hepatic expression of genes related to FA uptake and oxidation and very-low-density lipoprotein synthesis/export did not differ among the groups. However, the mRNA levels of sterol regulatory element-binding protein (SREBP)-1c and its downstream genes FA synthase, acetyl-coenzyme A carboxylase 1, and diacylglycerol acyltransferase 1 were inversely correlated with fibrosis stage. Immunoblot analysis revealed a remarkable reduction in mature SREBP-1c levels in advanced NASH. Furthermore, hepatic expression of tumor necrosis factor-alpha increased in accordance with fibrosis progression, which was possibly related to the decrease in hepatic SREBP-1c expression. CONCLUSIONS Down-regulation of SREBP-1c and lipogenic enzymes may be associated with the development of burned-out NASH.

Eicosapentaenoic acid improves hepatic steatosis independent of PPARα activation through inhibition of SREBP-1 maturation in mice

Eicosapentaenoic acid (EPA) in fish oil is known to improve hepatic steatosis. However, it remains unclear whether such action of EPA is actually caused by peroxisome proliferator-activated receptor α (PPARα) activation. To explore the contribution of PPARα to the effects of EPA itself, male wild-type and Ppara-null mice were fed a saturated fat diet for 16 weeks, and highly (>98%)-purified EPA was administered in the last 12 weeks. Furthermore, the changes caused by EPA treatment were compared to those elicited by fenofibrate (FF), a typical PPARα activator. A saturated fat diet caused macrovesicular steatosis in both genotypes. However, EPA ameliorated steatosis only in wild-type mice without PPARα activation, which was evidently different from numerous previous observations. Instead, EPA inhibited maturation of sterol-responsive element-binding protein (SREBP)-1 in the presence of PPARα through down-regulation of SREBP cleavage-activating protein and site-1 protease. Additionally, EPA suppressed fatty acid uptake and promoted hydrolysis of intrahepatic triglycerides in a PPARα-independent manner. These effects were distinct from those of fenofibrate. Although fenofibrate induced NAPDH oxidase and acyl-coenzyme A oxidase and significantly increased hepatic lipid peroxides, EPA caused PPARα-dependent induction of superoxide dismutases, probably contributing to a decrease in the lipid peroxides. These results firstly demonstrate detailed mechanisms of steatosis-ameliorating effects of EPA without PPARα activation and ensuing augmentation of hepatic oxidative stress.

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.

Purification and Identification of Antihypertensive Peptides from Fermented Buckwheat Sprouts

Buckwheat (Fagopyrum esculentum) is rich in antihypertensive compounds. This study investigated the effect of lactic-fermented buckwheat sprouts (neo-FBS) on level, identification, and potency of blood pressure-lowering (BPL) compounds. A single oral dose of 1.0 mg/kg body weight buckwheat sprouts (BS) in spontaneously hypertensive rats did not show significant BPL activity, whereas neo-FBS significantly decreased blood pressure. HPLC of neo-FBS identified two peaks absent in the profile of BS. The peak exhibiting potent BPL activity was fractionated, and six peptides (DVWY, FDART, FQ, VAE, VVG, and WTFR) and tyrosine were identified by LC-MS/MS and Edman degradation. Single oral dose administration of the peptides revealed significant BPL effect of all the peptides, with the most potent being DVWY, FQ, and VVG. DVWY, VAE, and WTFR are novel. This study demonstrates that lactic fermentation of BS produces new, highly potent antihypertensive peptides and increases active compounds GABA and tyrosine already present in BS.

Steatogenesis in adult-onset type II citrullinemia is associated with down-regulation of PPARα

SLC25A13 (citrin or aspartate-glutamate carrier 2) is located in the mitochondrial membrane in the liver and its genetic deficiency causes adult-onset type II citrullinemia (CTLN2). CTLN2 is one of the urea cycle disorders characterized by sudden-onset hyperammonemia due to reduced argininosuccinate synthase activity. This disorder is frequently accompanied with hepatosteatosis in the absence of obesity and ethanol consumption. However, the precise mechanism of steatogenesis remains unclear. The expression of genes associated with fatty acid (FA) and triglyceride (TG) metabolism was examined using liver samples obtained from 16 CTLN2 patients and compared with 7 healthy individuals. Although expression of hepatic genes associated with lipogenesis and TG hydrolysis was not changed, the mRNAs encoding enzymes/proteins involved in FA oxidation (carnitine palmitoyl-CoA transferase 1α, medium- and very-long-chain acyl-CoA dehydrogenases, and acyl-CoA oxidase 1), very-low-density lipoprotein secretion (microsomal TG transfer protein), and FA transport (CD36 and FA-binding protein 1), were markedly suppressed in CTLN2 patients. Serum concentrations of ketone bodies were also decreased in these patients, suggesting reduced mitochondrial β-oxidation activity. Consistent with these findings, the expression of peroxisome proliferator-activated receptor α (PPARα), a master regulator of hepatic lipid metabolism, was significantly down-regulated. Hepatic PPARα expression was inversely correlated with severity of steatosis and circulating ammonia and citrulline levels. Additionally, phosphorylation of c-Jun-N-terminal kinase was enhanced in CTLN2 livers, which was likely associated with lower hepatic PPARα. Collectively, down-regulation of PPARα is associated with steatogenesis in CTLN2 patients. These findings provide a novel link between urea cycle disorder, lipid metabolism, and PPARα.

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.

Multiple roles of PPARα in brown adipose tissue under constitutive and cold conditions

Peroxisome proliferator‐activated receptor α (PPARα) is a member of the nuclear receptor family, regulating fatty acid degradation in many organs. Two‐dimensional SDS‐PAGE of brown adipose tissue (BAT) from PPARα‐null mice produced a higher‐density spot. Proteomic analysis indicated that the protein was pyruvate dehydrogenase β (PDHβ). To observe PDHβ regulation in BAT, the organ was stimulated by long‐term cold exposure, and the activities of associated enzymes were investigated. Histological and biochemical analyses of BAT showed a significant decrease in the triglyceride content in wild‐type mice and some degree of decrease in PPARα‐null mice on cold exposure. Analyses of molecules related to glucose metabolism showed that the expression of PDHβ is under PPARα‐specific regulation, and that glucose degradation ability may decrease on cold exposure. In contrast, analyses of molecules related to fatty acid metabolism showed that numerous PPARα/γ target molecules are induced on cold exposure, and that fatty acid degradation ability in wild‐type mice is markedly enhanced and also increases to same degree in PPARα‐null mice on cold exposure. Thus, this study proposes novel and multiple roles of PPARα in BAT.

Pretreatment by low-dose fibrates protects against acute free fatty acid-induced renal tubule toxicity by counteracting PPARα deterioration.

Development of a preventive strategy against tubular damage associated with proteinuria is of great importance. Recently, free fatty acid (FFA) toxicities accompanying proteinuria were found to be a main cause of tubular damage, which was aggravated by insufficiency of peroxisome proliferator-activated receptor alpha (PPARα), suggesting the benefit of PPARα activation. However, an earlier study using a murine acute tubular injury model, FFA-overload nephropathy, demonstrated that high-dose treatment of PPARα agonist (0.5% clofibrate diet) aggravated the tubular damage as a consequence of excess serum accumulation of clofibrate metabolites due to decreased kidney elimination. To induce the renoprotective effects of PPARα agonists without drug accumulation, we tried a pretreatment study using low-dose clofibrate (0.1% clofibrate diet) using the same murine model. Low-dose clofibrate pretreatment prevented acute tubular injuries without accumulation of its metabolites. The tubular protective effects appeared to be associated with the counteraction of PPARα deterioration, resulting in the decrease of FFAs influx to the kidney, maintenance of fatty acid oxidation, diminution of intracellular accumulation of undigested FFAs, and attenuation of disease developmental factors including oxidative stress, apoptosis, and NFκB activation. These effects are common to other fibrates and dependent on PPARα function. Interestingly, however, clofibrate pretreatment also exerted PPARα-independent tubular toxicities in PPARα-null mice with FFA-overload nephropathy. The favorable properties of fibrates are evident when PPARα-dependent tubular protective effects outweigh their PPARα-independent tubular toxicities. This delicate balance seems to be easily affected by the drug dose. It will be important to establish the appropriate dosage of fibrates for treatment against kidney disease and to develop a novel PPARα activator that has a steady serum concentration regardless of kidney dysfunction.

Effect of bezafibrate on hepatic oxidative stress: comparison between conventional experimental doses and clinically-relevant doses in mice.

Abstract Several rodent studies have demonstrated that fibrate drugs can activate peroxisome proliferator-activated receptor α (PPARα) and increase reactive oxygen species (ROS) production. The persistence of strong PPARα activation is considered to be a possible mechanism related to the adverse effects of these agents in humans. We recently found that bezafibrate-treated mice at clinically-relevant doses (10 mg/kg/day) exhibited similar pharmacokinetics to humans, but were different from previous rodent data (> 50 mg/kg/day). To examine whether clinical doses of bezafibrate do in fact activate PPARα and increase hepatic oxidative stress in mice, we administered bezafibrate to wild-type and Ppara-null mice at high (100 mg/kg/day) or low (10 mg/kg/day) doses and assessed ROS-related pathways in the liver. High-dose bezafibrate increased hepatic lipid peroxides in a PPARα-dependent manner, likely from discordant induction of PPARα-regulated ROS-generating enzymes (acyl-CoA oxidase, cytochrome P450 4A, and NADPH oxidase) and enhancement of mitochondrial β-oxidation. The treatment also activated protein kinase C and phosphatidylinositol-3-kinase in wild-type mice only, suggesting an association between strong PPARα activation and an altered cell signaling cascade. Meanwhile, low-dose bezafibrate reduced serum/liver triglycerides in both genotypes without activating PPARα or enhancing hepatic oxidative stress. These results may support the safety of bezafibrate treatment at clinically-relevant doses.