Reduction in Secretion of Very Low Density Lipoprotein-Triacylglycerol by a Matrix Metalloproteinase Inhibitor in a Rat Model of Diet-Induced Hypertriglyceridemia.

Matrix metalloproteinase inhibitors (MMPIs) reduced serum triacylglycerol (TAG) levels in streptozotocin-induced diabetic rats and Zucker fa/fa rats in our previous study. However, the mechanisms underlying TAG reduction by MMPIs remain unclear. The present study aimed to elucidate the mechanism by which F81-1144b, an MMPI, lowers serum TAG levels in an animal model of high-sucrose diet (HSD)-induced hypertriglyceridemia. F81-1144b was repeatedly administered to rats fed HSD, and its effects were evaluated on TAG levels in serum and the liver, very low density lipoprotein (VLDL) secretion, de novo fatty acid (FA) synthesis in the liver, and the expression of genes regulating the metabolism of FA, TAG, and VLDL in the liver and serum. F81-1144b lowered TAG levels in serum and the liver, VLDL-TAG secretion, de novo FA synthesis in the liver, and serum levels of insulin and glucose. F81-1144b suppressed the expression of genes related to the de novo synthesis of FA and TAG, key proteins (lipin 1 and apolipoprotein CIII) responsible for VLDL metabolism, and sterol regulatory element-binding protein-1c and carbohydrate response element-binding protein. F81-1144b little affected the expression of genes related directly to the degradation of TAG or FA, but it upregulated that of gene for uncoupling protein 2 in the liver. These results suggest that MMPIs are a novel type of therapeutic agent for the treatment of hypertriglyceridemia, because the metabolic effects of F81-1144b expected from changes in the expression of genes regulating lipid metabolism would alter metabolism differently from those induced by fibrates, niacin, or n-3 FAs.

Fatty Acid ß-Oxidation Plays a Key Role in Regulating cis-Palmitoleic Acid Levels in the Liver.

Different monounsaturated fatty acid (MUFA) species have distinct pathophysiological activities. cis-Palmitoleic acid (16:1n-7) was previously reported to improve insulin sensitivity in animal studies. The proportions of hepatic MUFAs are generally considered to reflect changes in the activities of fatty acid modifications (∆9 desaturation and fatty acid elongation). However, hepatic levels of 16:1n-7 are markedly lower than those of oleic acid (18:1n-9). Nevertheless, no convincing explanation has yet been provided for the low level of 16:1n-7. We hypothesized that fatty acid degradation plays a key role in maintaining a low 16:1n-7 proportion in the liver. In order to corroborate the link between ß-oxidation and the proportion of 16:1n-7, rats were fed a control diet, fed a fat-free diet to up-regulate fatty acid modifications, but not ß-oxidation, or treated with clofibric acid to up-regulate fatty acid modifications and ß-oxidation. The nutritional manipulation markedly increased the proportions of 16:1n-7, 18:1n-9, and cis-vaccenic acid (18:1n-7). Although the pharmacological manipulation enhanced fatty acid modifications to largely the same extent as the nutritional manipulation and markedly elevated the proportion of 18:1n-9, those of 16:1n-7 and 18:1n-7 remained largely unchanged. The oxidation rates of 16:1n-7, 18:1n-9, and 18:1n-7 in liver slices were in the following order: 16:1n-7>18:1n-7≑18:1n-9 in control livers, and were increased by the pharmacological manipulation and decreased by the nutritional manipulation. These results strongly suggest that ß-oxidation, in concert with fatty acid modifications, plays a key role in regulating the MUFA profile and is crucially involved in maintaining low 16:1n-7 levels in the liver.

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis.

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

A simple and sensitive method for the determination of fibric acids in the liver by liquid chromatography.

Fibrates are used in biochemical and pharmacological studies as bioactive tools. Nevertheless, most studies have lacked information concerning the concentrations of fibric acids working inside tissues because a simple and sensitive method is not available for their quantitation. This study aimed to develop a simple and sensitive bioanalytical method for the quantitation of clofibric, bezafibric and fenofibric acids in samples of very small portions of tissues. Fibric acids were extracted into n-hexane-ethyl acetate from tissue homogenates (10 mg of liver, kidney or muscle) or serum (100 µL) and were derivatized with 4-bromomethyl-6,7-dimethoxycoumarin, followed by HPLC with fluorescence detection. These compounds were separated isocratically on a reversed phase with acetonitrile-water. Standard analytical curves were linear over the concentration range of 0.2-20 nmol/10 mg of liver. Precision and accuracy were within acceptable limits. Recovery from liver homogenates ranged from 93.03 to 112.29%. This method enabled the quantitation of fibric acids in 10 mg of liver from rats treated with clofibric acid, bezafibric acid or fenofibrate. From these analytical data, it became clear that there was no large difference in ratio of acyl-CoA oxidase 1 (Acox1) mRNA level to fibric acid content in the liver among the three fibric acids, suggesting that these three fibric acids have similar potency to increase expression of the Acox1 gene, which is a target of peroxisome proliferator-activated receptor α. Thus, the proposed method is a simple, sensitive and reliable tool for the quantitation of fibric acids working in vivo inside livers.

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy.

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.

Fibrates reduce triacylglycerol content by upregulating adipose triglyceride lipase in the liver of rats.

Hepatic triacylglycerol (TAG) homeostasis is maintained by carefully regulated balance between its synthesis and disposal. Impairment in this balance causes steatosis. The aims of this study were i) to uncover whether fibrates control TAG concentration through the action of adipose triglyceride lipase (ATGL) and ii) to compare the potency of the effects on ATGL expression and TAG concentration among fenofibrate, bezafibrate, and clofibric acid in the liver of rats. Treatments of rats with the three fibrates induced ATGL and concomitantly decreased hepatic TAG concentration. The upregulation of ATGL was likely mediated through the activation of peroxisome proliferator-activated receptor α. Fibrates also expanded capacity of fatty acid ß-oxidation. Importantly, three fibric acids (fenofibric, bezafibric, and clofibric acids) that are active metabolites formed in the liver exhibited almost the same potency to elevate ATGL expression in vivo, despite the fact that there were considerable differences in this regard among fenofibrate, bezafibrate, and clofibric acid when compared on the basis of their dosage. These results suggest that ATGL represents a potential therapeutic target for ameliorating hepatic steatosis and that fibric acids are promising agents to ameliorate and/or protect against hepatic steatosis.

Abnormalities in the Metabolism of Fatty Acids and Triacylglycerols in the Liver of the Goto-Kakizaki Rat: A Model for Non-Obese Type 2 Diabetes.

The Goto-Kakizaki (GK) rat is widely used as an animal model for spontaneous-onset type 2 diabetes without obesity; nevertheless, little information is available on the metabolism of fatty acids and triacylglycerols (TAG) in their livers. We investigated the mechanisms underlying the alterations in the metabolism of fatty acids and TAG in their livers, in comparison with Zucker (fa/fa) rats, which are obese and insulin resistant. Lipid profiles, the expression of genes for enzymes and proteins related to the metabolism of fatty acid and TAG, de novo synthesis of fatty acids and TAG in vivo, fatty acid synthase activity in vitro, fatty acid oxidation in liver slices, and very-low-density-lipoprotein (VLDL)-TAG secretion in vivo were estimated. Our results revealed that (1) the TAG accumulation was moderate, (2) the de novo fatty acid synthesis was increased by upregulation of fatty acid synthase in a post-transcriptional manner, (3) fatty acid oxidation was also augmented through the induction of carnitine palmitoyltransferase 1a, and (4) the secretion rate of VLDL-TAG remained unchanged in the livers of GK rats. These results suggest that, despite the fact that GK rats exhibit non-obese type 2 diabetes, the upregulation of de novo lipogenesis is largely compensated by the upregulation of fatty acid oxidation, resulting in only moderate increase in TAG accumulation in the liver.

Up-regulation of stearoyl-CoA desaturase 1 increases liver MUFA content in obese Zucker but not Goto-Kakizaki rats.

The Goto-Kakizaki (GK) rat is an animal model for spontaneous-onset, non-obese type 2 diabetes. Despite abundant evidence about disorders in metabolism, little information is available about fatty acid metabolism in the liver of GK rats. This study aimed to investigate the characteristics of the fatty acid profile, particularly MUFA, and the mechanism underlying the alterations in fatty acid profiles in the liver of GK rats. The activities of enzymes that participate in the biosynthesis of MUFA, expressions of genes encoding these enzymes, and the fatty acid profile in the liver were compared with those of obese Zucker (fa/fa) (ZF) rats, which are obese and non-diabetic. Stearoyl-CoA desaturase (SCD) activity and SCD1 gene expression were considerably up-regulated in GK rats, and these levels were largely comparable to those in ZF rats. However, the proportions and contents of oleic acid and palmitoleic acid were very low considering the highly elevated activity of SCD in the liver of GK rats, when compared with ZF rats. Palmitoyl-CoA chain elongation (PCE) activity and fatty acid elongase (Elovl6) gene expression were markedly up-regulated in ZF rats, whereas PCE activity was up-regulated much less and Elovl6 gene expression was unchanged in GK rats. These results suggest the possibility that up-regulation of gene expression of Elovl6 along with SCD1 is indispensable to elevate the proportions and contents of oleic acid in the liver.