Abstract
Abstract
Phytanic acid (PA) is a fatty acid (FA) that is present in ruminant products, and a natural agonist of the peroxisome proliferator-activated receptors (PPAR). Because the PPARs are pivotal in the regulation of glucose and FA homeostasis, it has been suggested that PA could positively regulate these processes, in which case, it could be considered as a bioactive compound with health-improving properties that can be used to fight against the metabolic syndrome (MS). The objectives of this PhD study were to determine the impact of total PA on glucose uptake and FA β-oxidation in skeletal muscles, and to elucidate the total content of PA and the distribution of its diastereomers in milk as affected by feed composition.
In this project, we established primary porcine myotubes as an efficient skeletal muscle model for metabolic studies. Satellite cells (SC) derived from porcine muscles were cultured to generate differentiated primary porcine myotubes. Viability studies were performed to determine which concentrations or length of treatments could be tolerated by the myotubes under glucose uptake, glycogen synthesis, and FA oxidation (FAO) experiments. Optimization of glucose uptake assay using cytochalasin B revealed that both the insulin-mediated and non-insulin mediated mechanisms of glucose uptake were functioning in the myotubes. Exposures to myotubes of excess glucose during the analysis of glucose uptake, and palmitate during the analysis of acylcarnitine, rendered the myotubes insulin resistant and inhibited the oxidation of palmitoylcarnitine (C16), phenomena of which can be expressed by skeletal muscles.
During the elucidation of the metabolic impact of PA, glucose uptake, glycogen synthesis and FAO were analyzed by the use of tritiated 2-deoxyglucose, 14C- glucose and 13C-palmitate, respectively. It was shown that physiological amounts of PA, which included 10 µM, enhanced glucose uptake, especially at low concentrations of insulin, but PA could not activate the incorporation of glucose into glycogen, except in the presence of insulin. Thus, PA seemed to regulate glucose uptake in both an insulin-dependent and insulin-independent fashion. When the myotubes were rendered insulin resistant by exposure to excess glucose, it was neither possible for PA nor insulin to stimulate glucose uptake or glycogen synthesis. During the analysis of β-oxidation using acylcarnitine profiling, we could show that PA enhanced the β-oxidation flux in the myotubes, as it caused an increase in the content of acetylcarnitine (C2) and a decrease in the C16/C2 ratio. Regarding stimulation of β-oxidation also 10 µM PA was an effective dose. However, we could not conclude if the induction in FAO by PA was through the induction of PPARα, since a PPARα agonist was necessary as a control to validate the changes observed.
It has previously been shown that cow breed and feed composition affect milk-output and FA composition, respectively, and that green feed increases the content of PA in milk-fat. In this project, milk was sampled from grazing Danish Holstein and Danish Jersey cows May and September periods, and the total content of PA and its diastereomers (RRR PA and SRR PA) in the milk-fat of the cows were studied by gas chromatography-mass-spectrometry analysis. The milk yield was higher in the Danish Holstein than the Danish Jersey cows, but the breed did not affect the total content of PA in milk-fat. The total content of PA was higher during the grazing period of September than in May. However, differences were small and the intake of green feed could not be related positively to the total content of PA. The distribution of the diastereomers was affected by feeding, as the content of the RRR PA was positively related to the intake of grazed legumes. This finding indicates that it is possible to manipulate the PA isomer distribution through strategic feeding.
In conclusion, a concentration of 10 µM PA achievable through dietary intake, is an acceptable amount in in vitro studies, and can stimulate both glucose uptake and FAO. The mechanisms behind these inductions need subsequent elucidations.
Phytanic acid (PA) is a fatty acid (FA) that is present in ruminant products, and a natural agonist of the peroxisome proliferator-activated receptors (PPAR). Because the PPARs are pivotal in the regulation of glucose and FA homeostasis, it has been suggested that PA could positively regulate these processes, in which case, it could be considered as a bioactive compound with health-improving properties that can be used to fight against the metabolic syndrome (MS). The objectives of this PhD study were to determine the impact of total PA on glucose uptake and FA β-oxidation in skeletal muscles, and to elucidate the total content of PA and the distribution of its diastereomers in milk as affected by feed composition.
In this project, we established primary porcine myotubes as an efficient skeletal muscle model for metabolic studies. Satellite cells (SC) derived from porcine muscles were cultured to generate differentiated primary porcine myotubes. Viability studies were performed to determine which concentrations or length of treatments could be tolerated by the myotubes under glucose uptake, glycogen synthesis, and FA oxidation (FAO) experiments. Optimization of glucose uptake assay using cytochalasin B revealed that both the insulin-mediated and non-insulin mediated mechanisms of glucose uptake were functioning in the myotubes. Exposures to myotubes of excess glucose during the analysis of glucose uptake, and palmitate during the analysis of acylcarnitine, rendered the myotubes insulin resistant and inhibited the oxidation of palmitoylcarnitine (C16), phenomena of which can be expressed by skeletal muscles.
During the elucidation of the metabolic impact of PA, glucose uptake, glycogen synthesis and FAO were analyzed by the use of tritiated 2-deoxyglucose, 14C- glucose and 13C-palmitate, respectively. It was shown that physiological amounts of PA, which included 10 µM, enhanced glucose uptake, especially at low concentrations of insulin, but PA could not activate the incorporation of glucose into glycogen, except in the presence of insulin. Thus, PA seemed to regulate glucose uptake in both an insulin-dependent and insulin-independent fashion. When the myotubes were rendered insulin resistant by exposure to excess glucose, it was neither possible for PA nor insulin to stimulate glucose uptake or glycogen synthesis. During the analysis of β-oxidation using acylcarnitine profiling, we could show that PA enhanced the β-oxidation flux in the myotubes, as it caused an increase in the content of acetylcarnitine (C2) and a decrease in the C16/C2 ratio. Regarding stimulation of β-oxidation also 10 µM PA was an effective dose. However, we could not conclude if the induction in FAO by PA was through the induction of PPARα, since a PPARα agonist was necessary as a control to validate the changes observed.
It has previously been shown that cow breed and feed composition affect milk-output and FA composition, respectively, and that green feed increases the content of PA in milk-fat. In this project, milk was sampled from grazing Danish Holstein and Danish Jersey cows May and September periods, and the total content of PA and its diastereomers (RRR PA and SRR PA) in the milk-fat of the cows were studied by gas chromatography-mass-spectrometry analysis. The milk yield was higher in the Danish Holstein than the Danish Jersey cows, but the breed did not affect the total content of PA in milk-fat. The total content of PA was higher during the grazing period of September than in May. However, differences were small and the intake of green feed could not be related positively to the total content of PA. The distribution of the diastereomers was affected by feeding, as the content of the RRR PA was positively related to the intake of grazed legumes. This finding indicates that it is possible to manipulate the PA isomer distribution through strategic feeding.
In conclusion, a concentration of 10 µM PA achievable through dietary intake, is an acceptable amount in in vitro studies, and can stimulate both glucose uptake and FAO. The mechanisms behind these inductions need subsequent elucidations.
| Original language | English |
|---|---|
| Publication status | Published - 30 Sept 2012 |
Keywords
- primary porcine myotubes
- phytanic acid
- phytanic acid diastereomers
- insulin
- palmitic acid
- glucose uptake
- fatty acid oxidation
- acylcarnitine
- pasture
- legumes