Juvenile Visceral Steatosis Mice Research Articles

PS-B08-7: L-CARNITINE AMELIORATES DIABETIC KIDNEY DISEASE BY ALLEVIATING MITOCHONDRIAL ABNORMALITY IN SDT-FATTY RATS

Background and Aims: Abnormal fatty acid metabolism is associated with the progression of diabetic kidney disease (DKD). Carnitine plays a central role in β;-oxidation and energy production by transporting long-chain fatty acids from the cytoplasm to the mitochondria. Recently, we found that significant ectopic fat was accumulated in the kidney tissue in DKD patients compared with those in minimal change kidney disease patients. Juvenile visceral steatosis (jvs) mice, described as an animal model for systemic carnitine deficiency, introduce a marked ectopic fat accumulation in the kidney. These findings suggest that abnormal carnitine dynamics in DKD cause ectopic fat accumulation due to abnormality of the fatty acid metabolism. However, the association between abnormalities of carnitine metabolism and the progression of DKD is unknown. Method: Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model was used. Unilateral nephrectomy was performed and 0.3% salt water was administered at 6 weeks of age, and created a renal dysfunction model in SDT fatty rat (DKD model). Age-matched male Sprague-Dawley (SD) rat was used as a control. Experiment 1: We compared 3 groups; SD rats (n = 8), SDT fatty rats (n = 8), DKD rats (n = 8). Experiment 2: We compared 3 groups; SD rats with sham operation (n = 8), DKD rats (n = 7), DKD rats treated with L-carnitine administration (0.75% L-carnitine diet) for 10 weeks (n = 6). All rats were sacrificed at 17 weeks old. Carnitine was measured by the enzyme cycling method. Acylcarnitine was analyzed by the tandem-mass spectrometry. Results: Plasma and renal free carnitine levels and short/middle-long chain acylcarnitine ratio were significantly decreased in DKD rats compared with those in SD and SDT fatty rats. The renal expression levels of organic cation / carnitine transporter 2 (OCTN2), carnitine palmitoyl transferase 1a (CPT1a), CPT2 and carnitine acetyltransferase (CrAT) were significantly decreased in DKD rats compared with those in SD and SDT fatty rats. Similarly, peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α) activity was decreased in DKD rats. L-carnitine administration significantly improved renal function, urinary albumin excretion, interstitial fibrosis and renal ectopic fat accumulation in DKD model rats. L-carnitine administration ameliorated the decrease of renal OCTN2, CPT1a, CPT2 and CrAT protein expression, and significantly improved PGC1α activity in DKD model rats. Furthermore, L-carnitine administration mitigated lipid peroxidation and mitochondrial dysfunction in DKD rats. Conclusion: L-carnitine administration might be a promising therapeutic strategy for improving renal function by alleviating mitochondrial dysfunction in DKD rat model.

POS-331 L-carnitine ameliorates diabetic kidney disease by alleviating mitochondrial abnormality and fatty acid accumulation in SDT-fatty rats

Abnormal fatty acid metabolism is associated with the progression of diabetic kidney disease (DKD). Carnitine plays a central role in fatty acid β-oxidation and energy production by transporting long-chain fatty acids from the cytoplasm to the mitochondria. Recently, we found that significant ectopic fat was accumulated in the kidney tissue in DKD patients compared with those in minimal change kidney disease patients independent of proteinuria levels, suggesting the abnormal carnitine dynamics in DKD. Juvenile visceral steatosis (jvs) mice, described as an animal model for systemic carnitine deficiency caused by a mutation in slc22a5 gene, introduce a marked ectopic fat accumulation in the kidney. These findings suggest that abnormal carnitine dynamics in DKD cause ectopic fat accumulation due to abnormality of the fatty acid metabolism. However, the association between abnormalities of carnitine metabolism and the progression of DKD is unknown. Therefore, we investigated the kinetics of carnitine and the effect of L-carnitine therapy in type 2 DKD model rats. Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model was used. Unilateral nephrectomy was performed and 0.3% salt water was administered at 6 weeks of age, and created a renal dysfunction model in SDT fatty rat (DKD model). Age-matched male Sprague-Dawley (SD) rat was used as a control. Experiment 1: We compared 3 groups; ①SD rats (n=8) ②SDT fatty rats (n=8) ③DKD rats (n=8). Experiment 2: We compared 3 groups; ①SD rats with sham operation (n=8) ②DKD rats (n=7) ③DKD rats treated with L-carnitine administration (0.75% L-carnitine diet) for 10 weeks (n=6). All rats were sacrificed at 17 weeks old. Carnitine was measured by the enzyme cycling method. Acylcarnitine was analyzed by the tandem-mass spectrometry. Plasma and renal free carnitine levels were significantly decreased and long-chain acylcarnitine levels were significantly increased in DKD rats compared with those in SD and SDT fatty rats. The renal expression levels of organic cation / carnitine transporter 2 (OCTN2), carnitine palmitoyl transferase 1a (CPT1a), CPT2 and carnitine acetyltransferase (CrAT) were significantly decreased in DKD rats compared with those in SD and SDT fatty rats. Similarly, acetyl-CoA carboxylase (ACC) activity was significantly increased and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α) and peroxisome proliferator-activated receptor α (PPARα) activity were decreased in DKD rats. Further, marked ectopic fat accumulation in the renal tubules and glomeruli in DKD model rats was observed. L-carnitine administration significantly improved renal function, urinary albumin excretion, interstitial fibrosis and renal ectopic fat accumulation in DKD model rats. L-carnitine administration ameliorated the decrease of renal OCTN2, CPT1a, CPT2 and CrAT protein expression, and significantly improved ACC, PGC1α, and PPARα activity in DKD model rats. Furthermore, L-carnitine administration mitigated lipid peroxidation and mitochondrial dysfunction in DKD rats Abnormal carnitine kinetics and dynamics might play an important role in the progression of DKD. L-carnitine administration might be a promising therapeutic strategy for improving renal function by alleviating mitochondrial dysfunction in DKD rat model.

Carnitine is necessary to maintain the phenotype and function of brown adipose tissue

The juvenile visceral steatosis (JVS) mouse is a mutant strain with an inherited systemic carnitine deficiency. Mice of this strain show clinical signs attributable to impaired heat production and disturbed energy production. Brown adipose tissue (BAT) is the primary site of non-shivering thermogenesis in the presence of uncoupling protein-1 (UCP-1) in rodents and humans, especially in infants. To investigate the possible cause of impaired heat production in BAT, we studied the morphological features, carnitine concentration, and UCP-1 production of BAT in JVS mice. The effect of carnitine administration on these parameters was also examined. JVS mice aged 5 or 10 days (60 each) and age-matched control mice were used in this study, along with 10-day-old JVS mice treated subcutaneously with L-carnitine once a day between postpartum days 5 and 10. JVS mice showed lower body temperatures and lower concentrations of carnitine in BAT. Morphologically, BAT cells in JVS mice contained large lipid vacuoles and small mitochondria, similar to those present in white adipose tissue cells. In addition, UCP-1 mRNA and protein expression levels were significantly reduced in JVS as compared with control mice. Carnitine treatment resulted in significant increases in body temperature and carnitine concentrations in BAT, together with the recovery of normal morphological features. UCP-1 mRNA and protein expression levels were also significantly increased. These findings strongly suggest that carnitine is essential for maintaining the function and morphology of BAT.

Induction of PDK4 in the heart muscle of JVS mice, an animal model of systemic carnitine deficiency, does not appear to reduce glucose utilization by the heart

Pyruvate dehydrogenase kinase 4 (PDK4) mRNA has been reported as an up-regulated gene in the heart and skeletal muscle of carnitine-deficient juvenile visceral steatosis (JVS) mice under fed conditions. PDK4 plays an important role in the inhibition of glucose oxidation via the phosphorylation of pyruvate dehydrogenase complex (PDC). This study evaluated the meaning of increased PDK4 mRNA in glucose metabolism by investigating PDK4 protein levels, PDC activity and glucose uptake by the heart and skeletal muscle of JVS mice. PDK4 protein levels in the heart and skeletal muscle of fed JVS mice were increased in accordance with mRNA levels, and protein was enriched in the mitochondria. PDK4 protein was co-fractionated with PDC in sucrose density gradient centrifugation, like PDK2 protein; however, the activities of the pyruvate dehydrogenase complex (PDC) active form in the heart and skeletal muscle of fed JVS mice were similar to those in fed control mice. Fed JVS mice showed significantly higher glucose uptake in the heart and similar uptake in the skeletal muscle compared with fed control mice. Thus, in carnitine deficiency under fed conditions, glucose was preferentially utilized in the heart as an energy source despite increased PDK4 protein levels in the mitochondria. The preferred glucose utilization may be involved in developing cardiac hypertrophy from carnitine deficiency in fatty acid oxidation abnormality.

Effect of gamma-butyrobetaine on fatty liver in juvenile visceral steatosis mice.

We pharmacokinetically examined the effect of gamma-butyrobetaine, a precursor of L-carnitine, on the change of fatty acid metabolism in juvenile visceral steatosis (JVS) mice, which have systemic L-carnitine deficiency due to lack of L-carnitine transporter activity. The concentrations of total free fatty acid (FFA), palmitic acid and stearic acid in the liver of JVS mice were significantly higher than those in wild-type mice. After intravenous administration of gamma-butyrobetaine (50 mg kg(-1)), the concentration of L-carnitine in the plasma of JVS mice reached about twice that of the control level and levels in the brain, liver and kidney were also significantly increased, whereas those in wild-type mice hardly changed. Although the plasma concentrations of FFA in both types of mice were unchanged after administration of gamma-butyrobetaine, the concentrations of palmitic acid and stearic acid were significantly decreased. In particular, the liver concentration of FFA in JVS mice was decreased to the wild-type control level, accompanied by significant decreases in long-chain fatty acids, palmitic acid and stearic acid, whereas those in wild-type mice were not changed. These results suggest that gamma-butyrobetaine can be taken up into organs, including the liver, of JVS mice, and transformed to L-carnitine. Consequently, administration of gamma-butyrobetaine may be more useful than that of L-carnitine itself for treatment of primary deficiency of carnitine due to a functional defect of the carnitine transporter.

Genetic deficiency of carnitine/organic cation transporter 2 (slc22a5) is associated with altered tissue distribution of its substrate pyrilamine in mice

Carnitine/organic cation transporter 2 (OCTN2) recognizes various cationic compounds as substrates in vitro, but information on its pharmacokinetic role in vivo is quite limited. This paper demonstrates altered tissue distribution of the OCTN2 substrate pyrilamine in juvenile visceral steatosis (jvs) mice, which have a hereditary defect of the octn2 gene. At 30 min after intravenous injection of pyrilamine, the tissue-to-plasma concentration ratio (K(p)) in the heart and pancreas was higher, whereas the K(p) in kidney and testis was lower in jvs mice compared with wild-type mice. Pyrilamine transport studies in isolated heart slices confirmed higher accumulation, together with lower efflux, of pyrilamine in the heart of jvs mice. The higher accumulation in heart slices of jvs mice was abolished by lowering the temperature, by increasing the substrate concentration, and in the presence of other H(1) antagonists or another OCTN2 substrate, carnitine, suggesting that OCTN2 extrudes pyrilamine from heart tissue. On the other hand, the lower distribution to the kidney of jvs mice was probably due to down-regulation of a basolateral transporter coupled with OCTN2, because, in jvs mice, (i) the K(p) of pyrilamine in kidney assessed immediately after intravenous injection (approximately 1 min) was also lower, (ii) the urinary excretion of pyrilamine was lower, and (iii) the uptake of pyrilamine in kidney slices was lower. The renal uptake of pyrilamine was saturable (K(m) approximately 236 microM) and was strongly inhibited by cyproheptadine, astemizole, ebastine and terfenadine. The present study thus indicates that genetic deficiency of octn2 alters pyrilamine disposition tissue-dependently.

Failure of the feeding response to fasting in carnitine-deficient juvenile visceral steatosis (JVS) mice: Involvement of defective acyl-ghrelin secretion and enhanced corticotropin-releasing factor signaling in the hypothalamus

Carnitine-deficient juvenile visceral steatosis (JVS) mice, suffering from fatty acid metabolism abnormalities, have reduced locomotor activity after fasting. We examined whether JVS mice exhibit specific defect in the feeding response to fasting, a key process of anti-famine homeostatic mechanism. Carnitine-deficient JVS mice showed grossly defective feeding response to 24 h-fasting, with almost no food intake in the first 4 h, in marked contrast to control animals. JVS mice also showed defective acyl-ghrelin response to fasting, less suppressed leptin, and seemingly normal corticotropin-releasing factor (CRF) expression in the hypothalamus despite markedly increased plasma corticosterone. The anorectic response was ameliorated by intraperitoneal administration of carnitine or acyl-ghrelin, with decreased CRF expression. Intracerebroventricular treatment of CRF type 2 receptor antagonist, anti-sauvagine-30, recovered the defective feeding response of 24 h-fasted JVS mice. The defective feeding response to fasting in carnitine-deficient JVS mice is due to the defective acyl-ghrelin and enhanced CRF signaling in the hypothalamus through fatty acid metabolism abnormalities. In this animal model, carnitine normalizes the feeding response through an inhibition of CRF.

Carnitine/Organic Cation Transporter OCTN2 (Slc22a5) Is Responsible for Renal Secretion of Cephaloridine in Mice

Carnitine/organic cation transporter (OCTN) 2 (SLC22A5) plays a pivotal role in renal tubular reabsorption of carnitine, a vitamin-like compound, on apical membranes of proximal tubules, but its role in relation to therapeutic drugs remains to be clarified. The purpose of the present study was to elucidate the involvement of OCTN2 in renal disposition of a beta-lactam antibiotic, cephaloridine (CER), based on experiments with juvenile visceral steatosis (jvs) mice, which have a functional deficiency of the octn2 gene. Renal clearance of CER during constant intravenous infusion in wild-type mice was much higher than could be accounted for by glomerular filtration, but was decreased by increasing the infusion rate with minimal change in kidney-to-plasma concentration ratio, suggesting the existence of saturable transport mechanism(s) across the apical membranes. The plasma concentration profile and kidney-to-plasma concentration ratio after intravenous injection in jvs mice were higher than those in wild-type mice, whereas renal clearance in jvs mice was much lower than that in wild-type mice and could be accounted for by glomerular filtration. Uptake of CER by mouse OCTN2 was shown in Xenopus laevis oocytes expressing mouse OCTN2. The CER transport by OCTN2 exhibited saturation with K(m) of approximately 3 mM, which is similar to the renal CER concentration exhibiting saturation in renal clearance in vivo. The OCTN2-mediated CER transport was inhibited by carnitine and independent of Na(+) replacement in the medium. These results show OCTN2 on apical membranes of proximal tubules plays a major role in renal secretion of CER in mice.

Effect of Carnitine Deprivation on Carnitine Homeostasis and Energy Metabolism in Mice with Systemic Carnitine Deficiency

Background/Aims: Juvenile visceral steatosis (jvs–/–) mice lack the activity of the carnitine transporter OCTN2 and are dependent on carnitine substitution. The effects of carnitine deprivation on carnitine homeostasis and energy metabolism are not known in jvs–/– mice. Methods: jvs–/– mice were studied 3, 6 and 10 days after carnitine deprivation, and compared to jvs–/– mice substituted with carnitine, wild-type (jvs+/+) and jvs+/– mice. Carnitine concentrations were assessed radioenzymatically. Results: Compared to wild-type mice, carnitine-treated jvs–/– mice had decreased plasma β-hydroxybutyrate levels and showed hepatic fat accumulation. The carnitine levels in plasma, liver and skeletal muscle were decreased by 58, 16 and 17%, respectively. After ten days of carnitine deprivation, the plasma carnitine concentration had fallen by 87% (to 2.3 µmol/l) and the tissue carnitine levels by ≈50% compared to carnitine-treated jvs–/– mice. Carnitine deprivation was associated with a further drop in plasma β-hydroxybutyrate and increased hepatic fat. Skeletal muscle glycogen stores decreased and lactate levels increased with carnitine deprivation, whereas tissue ATP levels were maintained. Conclusions: In jvs–/– mice, tissue carnitine stores are more resistant than carnitine plasma concentrations to carnitine deprivation. Metabolic changes (liver steatosis and loss of muscle glycogen stores) appear also early after carnitine deprivation.

Involvement of Carnitine/organic Cation Transporter OCTN2 (SLC22A5) in Distribution of its Substrate Carnitine to the Heart

This study was designed to clarify the pharmacological role of carnitine/organic cation transporter (Octn) family members in mouse heart. Immunohistochemical analysis revealed that Octn1 was exclusively expressed on endothelial cells in blood vessels. Octn2 was detected on the plasma membrane of cardiac muscle cells by immunoelectron microscopy. Octn3 was not detected in the heart. Integration plot analysis showed that coadministration of unlabeled L-carnitine reduced distribution of L-[3H]carnitine to the heart. L-[3H]Carnitine uptake in heart slices was reduced by carnitine analogs and various Octn2 substrates. L-[3H]Carnitine uptake by heart slices from juvenile visceral steatosis (jvs) mice, which have a hereditary octn2 gene deficiency, was negligible. Distribution of [3H]quinidine, another Octn2 substrate, to the heart was not reduced by L-carnitine, and [3H]quinidine uptake in heart slices was Na(-)-independent and inhibited by cationic drugs, but not carnitine analogs. [3H]Quinidine uptake by heart slices from jvs mice was similar to that of wild-type mice. These results demonstrate that OCTN2 is functionally expressed on the plasma membrane of muscle cells and is involved in distribution of carnitine to the heart. Some mechanism(s) other than OCTN2 is involved in the distribution of quinidine to the heart.

Spontaneous development of intestinal and colonic atrophy and inflammation in the carnitine-deficient jvs (OCTN2 −/−) mice

Carnitine is essential for transport of long-chain fatty acids into mitochondria for their subsequent β-oxidation, but its role in the gastrointestinal tract has not been well described. Recently several genetic epidemiologic studies have shown strong association between mutations in carnitine transporter genes OCTN1 and OCTN2 and a propensity to develop Crohn’s disease. This study aims to investigate role of carnitine and β-oxidation in the GI tract. We have studied the gastrointestinal tract effects of carnitine deficiency in a mouse model with loss-of-function mutation in the OCTN2 carnitine transporter. juvenile visceral steatosis (OCTN2 −/−) mouse spontaneously develops intestinal villous atrophy, breakdown and inflammation with intense lymphocytic and macrophage infiltration, leading to ulcer formation and gut perforation. There is increased apoptosis of jvs (OCTN2 −/−) gut epithelial cells. We observed an up-regulation of heat shock factor-1 (HSF-1) and several heat shock proteins (HSPs) which are known to regulate OCTN2 gene expression. Intestinal and colonic epithelial cells in wild type mice showed high expression and activity of the enzymes of β-oxidation pathway. These studies provide evidence of an obligatory role for carnitine in the maintenance of normal intestinal and colonic structure and morphology. Fatty acid oxidation, a metabolic pathway regulated by carnitine-dependent entry of long-chain fatty acids into mitochondrial matrix, is likely essential for normal gut function. Our studies suggest that carnitine supplementation, as a means of boosting fatty acid oxidation, may be therapeutically beneficial in patients with inflammation of the intestinal tract.

Pressure Overload-Induced Cardiomyopathy in Heterozygous Carrier Mice of Carnitine Transporter Gene Mutation

Primary systemic carnitine deficiency is an autosomal recessive disorder caused by a decreased renal reabsorption of carnitine because of mutations of the carnitine transporter OCTN2 gene, and hypertrophic cardiomyopathy is a common clinical feature of homozygotes. Although heterozygotes for OCTN2 mutations are generally healthy with normal cardiac performance, heterozygotes may be at risk for cardiomyopathy in the presence of additional risk factors, such as hypertension. To test this hypothesis, we investigated the effects of surgically induced pressure overload on the hearts of heterozygous mutants of a murine model of OCTN2 mutation, juvenile visceral steatosis mouse (jvs/+). Eleven-week-old jvs/+ mice and age-matched wild-type mice were used. At baseline, there were no differences in physical characteristics between wild-type and jvs/+ mice. However, plasma and myocardial total carnitine levels in jvs/+ mice were lower than in wild-type mice. Both wild-type and jvs/+ mice were subjected to ascending aortic constriction with or without 1% l-carnitine supplementation for 4 weeks. At 4 weeks after ascending aortic constriction, jvs/+ mice showed an exaggeration of cardiac hypertrophy and pulmonary congestion, further increased gene expression of atrial natriuretic peptide in the left ventricles, further deterioration of left ventricular fractional shortening, reduced myocardial phosphocreatine:adenosine triphosphate ratio, and increased mortality compared with wild-type mice; l-carnitine supplementation prevented these changes in jvs/+ mice subjected to ascending aortic constriction. In conclusion, cardiomyopathy and heart failure with energy depletion may be induced by pressure overload in heterozygotes for OCTN2 mutations and could be prevented by l-carnitine supplementation.

Prolonged effect of single carnitine administration on fasted carnitine-deficient JVS mice regarding their locomotor activity and energy expenditure

Carnitine is an essential cofactor for the oxidation of fatty acid in the mitochondria and an efficient therapeutics for primary carnitine deficiency. We herein analyzed the prolonged effects of carnitine on the reduced locomotor activity and energy metabolism of fasted carnitine-deficient juvenile visceral steatosis ( jvs − /− ) mice. We found that a single carnitine administration to 24-h fasted jvs − /− mice in the morning increased both the locomotor activity and oxygen consumption at night not only on the same day, but also on the next day, when the carnitine levels in the blood and tissues were already as low as at the original carnitine-deficient state. We also found that fat utilization for energy production significantly increased under fasting even in jvs − /− mice and was stimulated in the carnitine-administrated fasted jvs − /− mice at night, in comparison to that observed in the saline-administered jvs − /− mice, at least for 2 days even under the low plasma and tissue carnitine levels. These results suggest that the low tissue carnitine levels are therefore not the sole rate-limiting factor of general fatty acid oxidation in carnitine-deficient jvs − /− mice.

Organic Cation/Carnitine Transporter OCTN2 (Slc22a5) Is Responsible for Carnitine Transport across Apical Membranes of Small Intestinal Epithelial Cells in Mouse

The organic cation/carnitine transporter OCTN2 is responsible for renal tubular reabsorption of its endogenous substrate, carnitine, although its physiological role in small intestine remains controversial. Here we present direct evidence for a predominant role of OCTN2 in small intestinal absorption of carnitine based on experiments with juvenile visceral steatosis (jvs) mice, which have a hereditary deficiency of the octn2 gene. Uptake of carnitine, assessed with an Ussing-type chamber system, from the apical surface of the small intestine was saturable and higher than that from the basal surface in wild-type mice, whereas carnitine uptake having these characteristics was almost absent in jvs mice. Saturable uptake of carnitine was also confirmed in isolated enterocytes obtained from wild-type mice, and the Km value obtained (approximately 20 microM) was close to that reported for carnitine uptake by human embryonic kidney 293 cells stably expressing mouse OCTN2 (Slc22a5). The carnitine uptake by enterocytes was decreased in the presence of various types of organic cations, and this inhibition profile was similar to that of mouse OCTN2, whereas uptake of carnitine was quite small and unsaturable in enterocytes obtained from jvs mice. Immunohistochemical and immunoprecipitation analyses suggested colocalization of OCTN2 with PDZK1, an adaptor protein that functionally regulates OCTN2. Immunoelectron microscopy visualized both OCTN2 and PDZK1 in microvilli of absorptive epithelial cells. These findings indicate that OCTN2 is predominantly responsible for the uptake of carnitine from the apical surface of mouse small intestinal epithelial cells, and it may therefore be a promising target for oral delivery of therapeutic agents that are OCTN2 substrates.

Expression and distribution of OCTN2 in mouse epididymis and its association with obstructive azoospermia in juvenile visceral steatosis mice

L-carnitine, an essential cofactor for mitochondrial, beta-oxidation of long-chain fatty acids, is known to play important roles in sperm maturation and metabolism when spermatozoa pass and acquire motility in the epididymis. We reported that obstructive azoospermia occurred in the epididymis in the juvenile visceral steatosis (JVS) mice, which are OCTN2 dysfunction mice caused by mutations in the gene encoding OCTN2, have been used for animal models of primary systemic carnitine deficiency. The aim of present study is to investigate the expression of OCTN2 protein in the mouse epididymis and its relation between the localization of OCTN2 and obstructive azoospermia in JVS mice as animal models for human male infertility. Animals used in this study were wild-type (C57BL/6 J) mice (n = 4) and JVS mice (n = 4). We made a specific polyclonal antibody against OCTN2 and examined immunohistochemically the localization of OCTN2 in the mouse epididymis. OCTN2 was localized on the apical membrane of the principal cells of distal corpus and cauda epididymides. Immunocytochemistry demonstrated that OCTN2 was localized on the surface of microvillus upon the principal cells. In JVS mice, immunoreactivity started in a region immediately distal to where the sperm obstruction occurred. Our results suggest that OCTN2 functions as a carnitine transporter between the epithelium and the lumen in distal corpus and cauda epididymides and provides a clue as to why obstructive azoospermia is induced in distal parts of epididymis.

Fasting-induced reduction in locomotor activity and reduced response of orexin neurons in carnitine-deficient mice

We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis ( jvs −/−) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12 h/cycle), the nocturnal LA of jvs −/− mice was much less than the control ( jvs +/+ and jvs +/−) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs −/− mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs −/− mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs −/− mice. Sleep analysis revealed that fasted jvs −/− mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs +/+ mice. These results strongly suggest that the reduced LA in fasted jvs −/− mice is related to the inhibition of orexin neuronal activity.

Combined therapy with PPARα agonist and l-carnitine rescues lipotoxic cardiomyopathy due to systemic carnitine deficiency

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and are key regulators of fatty acid oxidation (FAO) in the heart. Systemic carnitine deficiency (SCD) causes disorders of FAO and induces hypertrophic cardiomyopathy with lipid accumulation. We hypothesized that activation of PPARalpha by fenofibrate, a PPARalpha agonist, in addition to conventional L-carnitine supplementation may exert beneficial effects on the lipotoxic cardiomyopathy in juvenile visceral steatosis (JVS) mouse, a murine model of SCD. Both wild-type (WT) and JVS mice were fed a normal chow, 0.2% fenofibrate containing chow (FE), a 0.1% L-carnitine containing chow (CA) or a 0.1% L-carnitine + 0.2% fenofibrate containing chow (CA + FE) from 4 weeks of age. Four to 8 animals per group were used for each experiment and 9 to 11 animals per group were used for survival analysis. At 8 weeks of age, JVS mice exhibited marked ventricular hypertrophy, which was more attenuated by CA + FE than by CA or FE alone. CA + FE markedly reduced the high plasma and myocardial triglyceride levels and increased the low myocardial ATP content to control levels in JVS mice. In JVS mice, myocardial 1,2-diacylglycerol (DAG) was significantly increased and showed a distinct fatty acid composition with elevation of 18:1(n-7,9) and 18:2(n-6) fatty acids compared with that in WT mice. CA + FE significantly altered the fatty acid composition of DAG and inhibited the membrane translocation of cardiac protein kinase C beta2 in JVS mice. Furthermore, CA + FE prevented the progressive left ventricular dysfunction and dramatically improved the survival rate in JVS mice (survival rate at 400 days after birth: 89 vs. 0%, P < 0.0001). PPARalpha activation, in addition to l-carnitine supplementation, may rescue the detrimental lipotoxic cardiomyopathy in SCD by improving cardiac energy and lipid metabolism as well as systemic lipid metabolism.

Attenuation of Cardiac Hypertrophy in Carnitine-Deficient Juvenile Visceral Steatosis (JVS) Mice Achieved by Lowering Dietary Lipid

We examined the development of cardiac hypertrophy in juvenile visceral steatosis (JVS) mice, a model of systemic carnitine deficiency, by varying the amount of lipid in the diet. Cardiac hypertrophy was markedly attenuated by decreasing soy bean oil (SBO) from 5% (w/w) to 1%. Triglyceride contents of the ventricles of JVS mice fed 1% SBO were significantly lower than in JVS mice fed 5% SBO. The addition of medium-chain triglycerides metabolically utilized by JVS mice did not affect the development of cardiac hypertrophy. On the other hand, the mRNA levels of atrial natriuretic peptide and skeletal alpha-actin, which are related to cardiac hypertrophy, were also attenuated by decreasing lipid in the diet. Adenylate energy charge and creatine phosphate in the heart of JVS mice at the early stage of hypertrophy were not significantly different from control mice given the same laboratory chow (4.6% of lipid). Although urinary prostaglandin F(2alpha) levels were found to be increased in JVS mice at 15 days of age when they developed cardiac hypertrophy, administration of aspirin was not efficacious. We, therefore, propose that the proportion of lipid in the diet is important in the development of cardiac hypertrophy in carnitine-deficient JVS mice, and that this is not related to prostaglandin formation.

Dietary fish oil attenuates cardiac hypertrophy in lipotoxic cardiomyopathy due to systemic carnitine deficiency

1,2-Diacylglycerol (DAG), a lipid second messenger that activates protein kinase C (PKC), is increased with a distinct fatty acid composition in the heart of the juvenile visceral steatosis (JVS) mouse, which develops pathological cardiac hypertrophy with lipid accumulation induced by the perturbation of fatty acid beta-oxidation due to systemic carnitine deficiency. Fish oil (FO) may exert its beneficial effects on the cardiomyopathy in JVS mice by modifying the molecular species composition of myocardial DAG. To test this hypothesis, we investigated the effects of dietary FO on the hypertrophied hearts in JVS mice. Both control and JVS mice were fed a FO diet (containing 10% FO) or a standard diet from 4 weeks of age. At 8 weeks of age, the ventricular-to-body weight ratio in JVS mice was 2.7-fold higher than that in controls (9.9 +/- 0.1 vs. 3.7 +/- 0.1 mg/g, P < 0.01) and was reduced by dietary FO (7.7 +/- 0.1 mg/g, P < 0.01 vs. JVS mice). In JVS mice, myocardial DAG levels were elevated by 2.3-fold with a distinct fatty acid composition with increases in C18:1n-7,9 and C18:2n-6 fatty acids compared with controls; dietary FO had no effects on the total DAG levels but significantly altered the fatty acid composition of DAG with reduction of both fatty acid species. Immunoblot analysis showed that dietary FO prevented the membrane translocation of cardiac PKCs alpha, beta2, and epsilon in JVS mice. Dietary FO did not affect the plasma and myocardial total carnitine levels in JVS mice. Furthermore, dietary FO significantly improved the progressive left ventricular dysfunction and survival rate in JVS mice. Dietary FO may attenuate cardiac hypertrophy with improvements in cardiac function and survival in JVS mice via modification of the molecular species composition of myocardial DAG and the consequent inhibition of PKC redistribution. These results suggest the implication of the molecular species composition of DAG in the pathogenesis of lipotoxic cardiomyopathy due to perturbations of fatty acid beta-oxidation.

Combined therapy with PPAR alpha agonist and carnitine supplementation rescues the detrimental cardiomyopathy with energy metabolism disorder

The juvenile visceral steatosis (JVS) mouse, a murine model of systemic carnitine deficiency (SCD), causes disorder of fatty acid oxidation and develops cardiac hypertrophy with lipid accumulation. We investigated the effect of fenofibrate, a PPARalpha agonist, in addition to carnitine supplementation on the hypertrophied heart in JVS mice. JVS mice were fed a normal chow, 0.1% carnitine/kg containing chow (CA), or 0.1%carnitine/kg and 0.2% fenofibrate/kg containing chow (CA + FE) from 4 weeks of age. At 8 weeks, the ventricular hypertrophy in JVS mice was more attenuated by CA + FE than by CA alone. CA + FE normalized the content of myocardial ATP and triglycerides to control levels, and prevented the deterioration of left ventricular function. Combined therapy with PPARalpha agonist and carnitine supplementation may be beneficial in treating the cardiomyopathy due to SCD. [Display omitted]