Alfred Lohninger

Dietary L-carnitine Stimulates Carnitine Acyltransferases in the Liver of Aged Rats

Aging affects oxidative metabolism in liver and other tissues. Carnitine acyltransferases are key enzymes of this process in mitochondria. As previously shown, the rate of transcription and activity of carnitine palmitoyltransferase CPT1 are also related to carnitine levels. In this study we compared the effect of dietary L-carnitine (100 mg L-carnitine/kg body weight/day over 3 months) on liver enzymes of aged rats (months 21–24) to adult animals (months 6–9) and age-related controls for both groups. The transcription rate of CPT1, CPT2, and carnitine acetyltransferase (CRAT) was determined by quantitative reverse transcription real-time PCR (RTQPCR) and compared to the activity of the CPT1A enzyme. The results showed that the transcription rates of CPT1, CPT2, and CRAT were similar in aged and adult control animals. Carnitine-fed old rats had a significant (p<0.05) 8–12-fold higher mean transcription rate of CPT1 and CRAT compared to aged controls, adult carnitine-fed animals, and adult controls, whereas the transcription rate of CPT2 was stimulated 2–3-fold in carnitine-fed animals of both age groups. With regard to the enzymatic activity of CPT1 there was a 1.5-fold increase in the old carnitine group compared to all other groups. RNA in situ hybridization also indicated an enhanced expression of CPT1A in hepatocytes from L-carnitine-supplemented animals. These results suggest that L-carnitine stimulates transcription of CPT1, CPT2, and CRAT as well as the enzyme activity of CPT1 in the livers of aged rats.

Non-invasive assessment of hepatic fat accumulation in chronic hepatitis C by 1H magnetic resonance spectroscopy.

BACKGROUND Liver biopsy is the standard method for diagnosis of hepatic steatosis, but is invasive and carries some risk of morbidity. AIMS AND METHODS Quantification of hepatocellular lipid content (HCL) with non-invasive single voxel (1)H magnetic resonance spectroscopy (MRS) at 3T was compared with histological grading and biochemical analysis of liver biopsies in 29 patients with chronic hepatitis C. Body mass index, indices of insulin resistance (homeostasis model assessment index, HOMA-IR), serum lipids and serum liver transaminases were also quantified. RESULTS HCL as assessed by (1)H MRS linearly correlated (r=0.70, p<0.001) with histological evaluation of liver biopsies and was in agreement with histological steatosis staging in 65% of the patients. Biochemically assessed hepatic triglyceride contents correlated with HCL measured with (1)H MRS (r=0.63, p<0.03) and allowed discriminating between none or mild steatosis versus moderate or severe steatosis. Patients infected with hepatitis C virus genotype 3 had a higher prevalence of steatosis (62%) which was not explained by differences in body mass or whole body insulin resistance. When these patients were excluded from correlation analysis, hepatic fat accumulation positively correlated with insulin resistance in the remaining hepatitis C patients (HCL vs. HOMA-IR, r=0.559, p<0.020, n=17). CONCLUSION Localized (1)H MRS is a valid and useful method for quantification of HCL content in patients with chronic hepatitis C and can be easily applied to non-invasively monitoring of steatosis during repeated follow-up measurements in a clinical setting.

A Combination of (ω–3) Polyunsaturated Fatty Acids, Polyphenols and L-Carnitine Reduces the Plasma Lipid Levels and Increases the Expression of Genes Involved in Fatty Acid Oxidation in Human Peripheral Blood Mononuclear Cells and HepG2 Cells

Background: Hyperlipidemia and obesity are associated with metabolic syndrome and increased risk in developing diabetes and cardiovascular disease. Nutritional supplements, e.g. L-carnitine and polyunsaturated fatty acids (PUFAs), exert lipid-lowering effects. Hence, the hypothesis that dietetic intervention reduces plasma lipid levels and metabolic enzymes in overweight hyperlipidemic subjects was tested. Subjects and Methods: In a prospective placebo-controlled double-blind study in 22 moderately hyperlipidemic obese humans consuming low-fat yoghurt enriched with a combination of low-dose PUFAs, polyphenols and L-carnitine (PPC) twice a day for 12 weeks were compared to 20 matching participants ingesting low-fat yoghurt. The effects on plasma lipids and expression of enzymes involved in regulation of fatty acid oxidation in peripheral blood mononuclear cells (PBMCs) and HepG2 cells were evaluated. Results: PPC consumption led to significantly reduced plasma free fatty acid (–29%) and triglyceride (–24%) concentrations (each p < 0.05). PPC application increased significantly peroxisome proliferator-activated receptor α (PPARα) mRNA abundances and those of PPARα target genes (carnitine palmitoyltransferases-1, CPT1A and CPT1B, carnitine acetyltransferase and organic cation transporter 2; each p < 0.05) in PBMCs. In controls, plasma lipid levels and PBMC gene expression did not change. These findings were substantiated by the results of cell culture experiments in HepG2 cells. Conclusion: Supplementation of PPC had marked lipid-lowering effects and PBMC gene expression profiles seemed to reflect nutrition-related metabolic changes.

Scavenger receptor, Class B, Type I provides an alternative means for β-VLDL uptake independent of the LDL receptor in tissue culture

Recent evidence suggests that scavenger receptor, class B, type I (SR-BI) plays a physiological role in VLDL metabolism. SR-BI was reported to mediate beta-VLDL uptake; however, cellular details of this process are not well characterized. In the present study we show that SR-BI delivers cholesterol derived from beta-VLDL to LDL receptor negative SR-BI over-expressing Chinese Hamster Ovarian cells (ldlA7-SRBI). Cell association of beta-VLDL was approximately 3 times higher after SR-BI over-expression, which was competed by beta-VLDL, but only to a lesser extent by HDL and LDL. Almost all of the associated beta-VLDL was located intracellularly, and therefore could not be released by a 50-fold excess of unlabeled beta-VLDL. beta-VLDL was degraded at a rate of 6 ng beta-VLDL/mg cell protein and hour. In contrast to ldlA7 cells, beta-VLDL association was competed by LDL in cells with a functional LDL receptor like CHO and HepG2 cells, indicating a strong impact of the LDL receptor in beta-VLDL uptake. beta-VLDL degradation was similar to ldlA7-SRBI cells. When beta-VLDL uptake was followed using fluorescence microscopy, beta-VLDL showed a different uptake pattern in SR-BI over-expressing cells, ldlA7-SRBI, compared to LDL receptor containing cells, CHO and HepG2.

A Chinese Hamster Ovarian Cell Line Imports Cholesterol by High Density Lipoprotein Degradation

Plasma high density lipoprotein (HDL) is inversely associated with the development of atherosclerosis. HDL exerts its atheroprotective role through involvement in reverse cholesterol transport in which HDL is loaded with cholesterol at the periphery and transports its lipid load back to the liver for disposal. In this pathway, HDL is not completely dismantled but only transfers its lipids to the cell. Here we present evidence that a Chinese hamster ovarian cell line (CHO7) adapted to grow in lipoprotein-deficient media degrades HDL and concomitantly internalizes HDL-derived cholesterol. Delivery of HDL cholesterol to the cell was demonstrated by a down-regulation of cholesterol biosynthesis, an increase in total cellular cholesterol content and by stimulation of cholesterol esterification after HDL treatment. This HDL degradation pathway is distinct from the low density lipoprotein (LDL) receptor pathway but also degrades LDL. 25-Hydroxycholesterol, a potent inhibitor of the LDL receptor pathway, down-regulated LDL degradation in CHO7 cells only in part and did not down-regulate HDL degradation. Dextran sulfate released HDL bound to the cell surface of CHO7 cells, and heparin treatment released protein(s) contributing to HDL degradation. The involvement of heparan sulfate proteoglycans and lipases in this HDL degradation was further tested by two inhibitors genistein and tetrahydrolipstatin. Both blocked HDL degradation significantly. Thus, we demonstrate that CHO7 cells degrade HDL and LDL to supply themselves with cholesterol via a novel degradation pathway. Interestingly, HDL degradation with similar properties was also observed in a human placental cell line.

Effects of Prenatal Treatment with Betamethasone, L-Carnitine, or Betamethasone-L-Carnitine Combinations on the Phosphatidylcholine Content and Composition of the Foetal and Maternal Rat Lung

Pregnant rats received 0.10 or 0.20 mg/kg body weight betamethasone, or 100 mg/kg body weight L-carnitine, or L-carnitine 100 mg/kg plus betamethasone 0.05 or 0.10 mg/kg body weight, or saline (controls) for three days before delivery of foetuses at day 19 of gestation. Dose-related effects on the dipalmitoyl phosphatidylcholine content and the phosphatidylcholine species composition of foetal and maternal lungs were determined. Betamethasone (0.10 and 0.20 mg/kg) or L-carnitine (100 mg/kg) significantly increased (p < 0.05) the dipalmitoyl phosphatidylcholine content in the foetal lungs, while only small changes were found in relative terms. Combinations of betamethasone (0.05 or 0.10 mg/kg) with L-carnitine (100 mg/kg) also significantly increased the dipalmitoyl phosphatidylcholine content of the foetal lungs above control values (p < 0.01) and above the values achieved with betamethasone alone (p < 0.05). In the maternal lungs a significant increase of the dipalmitoyl phosphatidylcholine content above the control values was only found after treatment with betamethasone-carnitine combinations, whereas compared with the foetal lung the relative increase of dipalmitoyl phosphatidylcholine as a fraction of total phosphatidylcholine was more pronounced after betamethasone treatment. The gas chromatographic method used separates two monoenoic phosphatidylcholine species with 32 carbon atoms in the acyl residues. These two phosphatidylcholine species showed striking differences between adult and foetal lungs. Palmitoleyl palmitoyl phosphatidylcholine predominates in the maternal lung, whereas palmitoyl palmitoleyl phosphatidylcholine is the major monoenoic phosphatidylcholine species with 32 carbon atoms in the foetal lung. These two species were not affected in maternal or foetal lung by betamethasone or L-carnitine treatment. In contrast, after treatment with betamethasone-carnitine combinations, a significant increase of the fraction of palmitoyl palmitoleyl phosphatidylcholine was found in foetal but not in the maternal lung. The results of the present study demonstrate that maternal glucocorticoid and carnitine treatment affects the maternal as well as the foetal lung but with different effects on the dipalmitoyl phosphatidylcholine content and phosphatidylcholine species composition.

Prenatal effects of betamethasone-L-carnitine combinations on fetal rat lung

Lungs of fetal rats between the 18th and 20th gestational day (total gestation lasting 22 days) were examined. There was a significant increase (p < 0.01) of the dipalmitoyl phosphatidylcholine content from day 19 to day 20 of gestation. In the second trial, pregnant rats were treated with different doses of betamethasone, L-carnitine, betamethasone-L-carnitine combinations, and saline (controls) for three days before Cesarean section on the 19th gestational day. Maternal injections of 0.10 mg/kg body weight betamethasone and 100 mg/kg body weight L-carnitine significantly (p < 0.05, p < 0.01 respectively) increased the dipalmitoyl phosphatidylcholine content of fetal lungs. Combinations of either 0.05 or 0.10 mg/kg body weight betamethasone with 100 mg L-carnitine also significantly increased the dipalmitoyl phosphatidylcholine content of the fetal lungs above control values (p < 0.001) and values achieved with betamethasone alone (p < 0.05). Maternal treatment with a betamethasone-L-carnitine combination on day 19 of gestation resulted in dipalmitoyl phosphatidylcholine levels comparable to those found on the 20th gestational day during normal lung maturation. Fetal rats delivered on the 20th gestational day survived, while fetuses delivered on the 19th gestational day did not survive.