Brita Ngum Che PhD Thesis 1 Health promoting factors from milk of cows fed green plant material - The role of phytanic acid Brita Ngum Che MSc in Molecular Biology Department of Food Science Faculty of Science and Technology Aarhus University Denmark A thesis submitted for the degree of Doctor of Philosophy at Aarhus University September 2012
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Health promoting factors from milk of cows fed green
plant material - The role of phytanic acid
Brita Ngum Che
MSc in Molecular Biology
Department of Food Science
Faculty of Science and Technology
Aarhus University
Denmark
A thesis submitted for the degree of Doctor of Philosophy at Aarhus University
September 2012
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Title of PhD project: Health promoting factors from milk of cows fed green plant
material - The role of phytanic acid
PhD student: Brita Ngum Che, MSc in Molecular Biology, Department of Food Science,
Faculty of Science and Technology, Aarhus University, Denmark
Supervisor: Associate professor Jette F. Young, Department of Food Science, Faculty of
Science and Technology, Aarhus University, Denmark
Co-supervisor: Assistant professor Mette K. Larsen, Department of Food Science,
Faculty of Science and Technology, Aarhus University, Denmark
Opponents:
1. Professor Karsten Kristiansen, Department of Biology, University of Copenhagen
2. Professor Ian Givens, Animal Science Research Group, University of Reding, United
Kingdom
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Preface
The work presented in the present PhD thesis titled “Health promoting factors from milk
of cows fed green plant material-the role of phytanic acid” centers around elucidating the
availability of phytanic acid in milk-fat and the relevance of this fatty acid on glucose and
fatty acid metabolism in humans. This work aimed at improving human health through the
differentiation of milk production enriched with phytanic acid.
This thesis is part of a “Green feed” project, where the metabolic impact of altered fatty
acid composition of milk-fat as affected by green feed was studied. I was specifically
interested in in vitro studies of glucose and fatty acid metabolism and the metabolic
impact of phytanic acid. The in vitro studies were conducted with a synthetic form of
phytanic acid, which is an isomeric mixture.
At the final stages of the project, the content of phytanic acid in milk-fat was accessed and
its diastereomers were observed to be differentially distribution under altered feeding
conditions. This was the reason behind the inclusion of the inclusion of studies on phytanic
acid diastereomers which was not part of the original setup of the project.
Regarding the setup of the thesis, an introduction to the thesis is presented, followed by a
literature review of factors that are relevant in understanding the work described in the
thesis. Results obtained from the study are discussed together, and some concluding
remarks are stated. Some future perspectives are included, and finally, an appendix of
manuscripts, which constitute work carried out in the project, is included.
This PhD project was based at the Department of Food Science at Aarhus University. Milk
analysis, in vitro cultures, and glucose metabolism studies were conducted at The
Department of Food Science, while the analyses of acylcarnitines were carried out at The
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Department of Forensic Science, in collaboration with The Research unit of Molecular
Medicine at Aarhus University.
The work presented in this PhD project was funded by The Danish Council for Strategic
Research and The Danish Cattle Federation, and was part of the scientific network called
“Tailored milk and human health”.
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Acknowledgements
I will commerce by expressing tremendous gratitude to my supervisor Jette F. Young,
Department of Food Science, for excellent supervision and guidance all through my PhD
studies. Thanks for the extreme understanding and support from you, especially during the
tough times. I am grateful for all the support right to the very last minute of completing my
thesis. To Lars I. Hellgren of The Technical University of Denmark, I am very grateful for
the fact that you conceptualized this project, thus making it at all possible. Your inputs at
every level have been indispensable for the realization of this thesis. Special thanks go to
my co-supervisor Mette K. Larsen, Department of Food Science, for her support both in
the laboratory and during the writing of manuscripts. I am grateful for the enthusiastic
discussion we have had concerning the milk-fat studies.
Special thanks to Niels Oksbjerg, Department of Food Science, for his contribution in the
isolation and culturing of porcine myotubes and the many discussions on glucose uptake
experiments and statistics. I will like to thank Jacob Holm Nielsen of Arla Foods, Braband,
Århus, for his fruitful input especially at the early stages of the project.
Special thanks to Mogens Johannsen and Rune Isaac Dupont Birkler at The Department of
Forensic Science at Aarhus University, for your help and support in the analysis of
acylcarnitines, and for you contribution in writing of manuscript. My gratitude also goes to
Niels Gregersen at The Research unit of Molecular Medicine at Aarhus University, thanks
for your inputs concerning the conception of acylcarnitine studies, but also for the warm
atmosphere and time spent at your laboratory.
Bente Andersen, Camilla Bjerg Kristensen, Caroline Nebel and Anne-Grethe Pedersen are
heartily thanked for contributing with excellent technical support.
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Aase Sørensen and Anne Hjorth Balling, special thank you for your patience and excellent
help in proof-reading my thesis and manuscripts
My office mates Sumangala Bhattacharya and Bjørn Nielsen, thanks for the many
interesting discussions, and a good working atmosphere. My colleagues in Foulum, thank
you for creating an interesting working environment and for your support in one way or
the other.
To my friend Ngonidzashe Chirinda, I very much appreciate the interesting discussions all
through the years in Foulum, and your suggestions regarding the structure of my thesis. To
my friend Steen Pedersen, thank you so much for excellent support in preparing some of
the figures for my thesis.
To my friend Bülent Kocaman, thank you for giving me the courage and support eight
years ago to quit my cleaning job and continue on my academic career. I am grateful to my
friend Marceline Pirkaniemi for her moral support. To Ina Lindgård and family, thank you
so much for being such a caring and supporting family. Your unlimited help with my kids
Nicole and Victoria has been invaluable and I will always be grateful for that.
Special thanks to my family for their moral support and up-backing, and to you Dad
thanks for implanting the will and zeal in me; you knew I would make it, and I am almost
there…..
Last but not the least, I am grateful for the tolerance, understand and love of my two
daughters Nicole and Victoria. It is incredibly cool of you two. You do not stop to amaze
me; first, you came into my life, then you gave me the chance to accomplish what I have
always dreamt of. For that, and all the unsaid, this work is dedicated to you Nicole and
Victoria.
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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
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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.
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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.
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Dansk Sammendrag
Fytansyre (PA) er en fedtsyre (FA) der findes i produkter fra drøvtyggere og er en naturlig
agonist for peroxisomere proliferator-aktiverede receptorer (PPAR). Fordi PPAR’erne er
centrale for reguleringen af glukose og FA homeostase, er det blevet foreslået, at PA kan
regulere disse processer positivt, og i så fald betragtes som en bioaktiv komponent med
sundhedsfremmende egenskaber, der vil kunne bruges i forbindelse med forebyggelse af
metabolisk syndrom (MS). Formålene med dette PhD-projekt har været at fastslå
betydningen af det totale PA på glukoseoptaget og FA β-oxidation i skeletmuskulatur, og
undersøge det totale indhold af PA og distributionen af dets diastereomerer i mælk
afhængig af fodersammensætning.
I dette projekt har vi etableret primære svine myotubes som en effektiv model for
skeletmuskulatur til metaboliske studier. Satellitceller (SC) fra muskelceller fra grise er
blevet dyrket for at etablere differentierede primære svine myotubes. Levedygtigheden af
myotubes blev undersøgt, for at bestemme hvilke koncentrationer og hvilke varigheder af
behandlinger som kunne tolereres af myotubes i forbindelse med glukoseoptag,
glykogensyntese og FA oxidations (FAO) eksperimenter. Optimering af glukoseoptag ved
brug af cytochalasin B viste, at både det insulinmedierede og ikke-insulinmedierede
glukoseoptag fungerede i myotubes. Overeksponering af glukose til Myotubes i forbindelse
med analysen af glukoseoptag, og palmitat i forbindelse med analyser af acylcarnitin,
gjorde myotubes insulinresistente og hæmmede oxidationen af palmitoylcarnitin (C16),
fænomener som er velkendte i skeletmuskulatur.
I forbindelse med undersøgelsen af PA’s metaboliske betydning, blev glukoseoptag,
glycogensyntese og FAO analyseret ved hjælp af henholdsvis tritieret 2-deoxyglukose, 14C-
glukose og 13C-palmitat. Det blev vist, at den fysiologiske mængde af PA, inklusiv 10 µM,
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forbedrede glukoseoptaget, især ved lave koncentrationer af insulin, men PA kunne ikke
aktivere dannelsen af glykogen fra glukose, medmindre der var insulin til stede. Dette
indikerer at PA regulerer glukoseoptag via både en insulin-afhængig og insulin-uafhængig
mekanisme. Når myotubes blev gjort insulinresistente ved overeksponering af glukose, var
det hverken muligt for PA eller insulin at stimulere glukoseoptag eller glykogensyntese. I
forbindelse med analysen af β-oxidation ved acylcarnitine profilering, kunne vi se, at PA
forbedrede β-oxidationen i myotubes, fordi det øgede indholdet af acetylcarnitin (C2) og
mindskede C16/C2 ratioen. Også i stimuleringen af β-oxidationen viste 10 µM PA sig at
være en effektiv dosis. Vi kunne dog ikke konkludere om PA-induceret FAO sket via
PPARα, fordi en veletableret PPARα agonisten ville være nødvendig som kontrol for at
kunne validere de ændringer, der blev observeret.
Det er tidligere vist, at kvægrace og fodersammensætning har betydning for såvel
mælkeudbytte som mælkens FA sammensætning, og at grønne fodermidler øger indholdet
af PA i mælkefedtet. I dette projekt blev der taget mælkeprøver fra græssende Holstein og
Jersey køer i maj og september, og det totale indhold af PA og dennes diastereomere (RRR
PA og SRR PA) i mælkefedtet fra køerne blev analyseret ved brug af gaschromatografi og
massespektrometri. Mælkeudbyttet var højere for Holstein end for Jersey køer, mens
racen ikke havde nogen betydning for det totale indhold af PA. Det totale indhold af PA var
højere i mælk fra september sammenlignet med maj. Forskellene var dog små, og det var
ikke muligt at relatere indtaget af grønne fodermidler positivt til mælkens PA indhold.
Fordelingen af diastereomererne var afhængig af fodringen, hvor indholdet af RRR PA var
positivt relateret til indtaget af bælgplanter i afgræsningen. Dette resultat indikerer, at det
er muligt at styrefordelingen af PA diastereomerer gennem strategisk fodring.
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Det kan konkluderes, at koncentrationen på 10 µM PA, der kan opnås via kosten, er en
acceptabel mængde i in vitro studier, og at dette kan stimulere både glukoseoptag og FAO.
Mekanismerne bag disse aktiveringer kræver dog nærmere undersøgelser.
PPARβ is also involved in FAO [136]. Recent findings about PPARβ show that it helps
against heat injury in fibroblasts by increasing cell proliferation [137]. It has also been
shown to improve myogenesis through the down-regulation of myostatin expression [138],
and it is involved in the prostaglandin-induced activation of blastocyst development in
mice [139]. Synthetic agonists of PPARβ include GW501516, which has been reported to
inhibit dyslipidemia, by slowing down the activity of cholesterol ester transfer protein, and
reducing the content of very low-density lipoprotein [140].
1.8.3 PPARγ
PPARγ is mainly expressed in adipose tissues but it is also found in other tissues such as
the liver [127]. The isoform exists in two forms (PPARγ1 and PPARγ2), and it is PPARγ2
that predominates in adipocytes [141]. It activates protein synthesis and adiponectin
secretion in adipocytes, and it is crucial for efficient white and brown adipocyte
differentiation [142]. PPARγ also regulates lipogenesis in the liver [143] and increases
insulin sensitivity in adipocytes, thus, ameliorating the dysfunction of fat cells in T2D
[144]. In muscles, PPARγ also increases insulin sensitivity and improves FAO [121, 145].
Targeting PPARγ for the modulation of IR is common [145, 146]. The thiazolidinedione
(TZDs) are well known synthetic ligands of PPARγ, and they are important in the
treatment of T2D [147]. Some mechanisms employed by TZDs in improving IR are by
stimulating the production of adiponectin, which suppresses gluconeogenesis, and
reducing the level of circulating free FAs through their esterification to glycerol [148, 149].
Nevertheless, PPARγ has shown to induce the production of adiposites [150, 151].
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1.9 Phytanic acid (PA)
1.9.1 Origin of PA and its diastereomers
The alcohol unit of chlorophyll; phytol, is the substrate for PA production (Figure 10). In
ruminants and in marine environments, oxidative processes of bacteria lead to the first of
the four steps in the degradation of chlorophyll; cleavage and oxidation of phytol from the
porphyrin ring of chlorophyll [155-157]. This step is the rate limiting step in chlorophyll
catabolism of chlorophyll and is controlled by the enzyme chlorophyllase [157]. The
generated phytol exists in two forms; E-phytol and Z-phytol, and when ingested by animals
or humans, can undergo a series of enzymatic reactions, including biohydrogenation, to
generate phytanic acid and afterwards, phytanoyl-CoA [158, 159], which can be
metabolized further. It was observed that E-phytol is the preferable substrate for the
PPAR-related metabolism of phytol [159, 160]. This conclusion was drawn from the fact
that E-phytol accumulates in the liver of PPARα-knockout mice fed phytol-rich diets [159].
More so, the enzymes responsible for phytol conversion to phytanoyl-CoA turned out to be
stereospecific [159]. It has also been shown that the aerobic and anaerobic activity of
bacteria on phytol generate both E- and Z-phytenic acid [161]. Thus, the role of bacteria in
the degradation of phytol is crucial, as these bacteria possess enzymes that are required for
the decarboxymethylation and oxidation of methyl-branched metabolites, without the
need of oxygen or the enzymes normally involved in methyl-branched FAs [162].
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Figure 10: Production of PA from phytol (Adopted with modifications from [158])
The generation of Z- and E-phytol from chlorophyll requires bacterial action. Both forms of phytol
can be differentially processed to phytenic acid, which in turn, is converted to either RRR PA or
SRR PA by an enoyl-CoA reductase. The esterification of both forms of PA generates RRR- and
SRR-phytanoyl-CoA.
PA is 16 C- long and has four methyl groups positioned at carbons 3, 7, 11 and 15, with the
carbon 3, 7 and 11 being chiral centers. Carbons 7 and 11 have R configurations, just like in
phytol, while carbon 3 is either R- or S- configured owing to the biohydrogenation of
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oxidized phytol. For this reason, PA occurs naturally as two diastereomers; SRR PA and
RRR PA [163]; see Figure 10. The SRR form has been shown to predominate in marine
animals while some terrestrial mammals have more of the RRR form [163-165]. Little is
known about the production and distribution of the two forms of PA in ruminal products
or about their physiological role. PA can be stored in the phospholipid and neutral
fractions of lipids in tissues and in milk [166]. Since humans lack the ability to cleave
phytol from chlorophyll [167], PA can only be obtained through the consumption of
ruminant or marine products [168, 169]. Differential deposition in lipid fractions of tissues
and metabolic rates of the diastereomers insinuates possible differences in the biological
activities of the two natural diastereomers of PA in humans [166, 170, 171].
1.9.2 Metabolism of PA in humans
After a phytol-rich meal, phytol is usually transported to the liver for catabolism, where
phytenic, phytanic and pristanic acids are formed [160]. Free phytol or PA taken up from
the diet can be converted into phytanoyl-CoA by the enzyme phytanoyl-CoA hydroxylase,
and further esterified and stored in lipids, hydrolyzed back to PA or used as a substrate for
alpha (α)- oxidation [172, 173]. Phytanoyl-CoA can also come directly from the diet, since it
is produced in ruminants. β-oxidation of PA is not directly possible due to the presence of
a methyl group on carbon 3 of PA ( see structure of PA in Figure 10).
The α- oxidation of PA occurs in the peroxisome [174] and the enzymes involved in the
process are illustrated in Figure 11. The actions of the hydroxylase and lyase enzymes on
phytanoyl-CoA and 2-hydroxylphytanoyl-CoA, respectively, are involved to produce
pristanic acid, with the release of formic acid as the primary product of α- oxidation [173,
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175, 176]. Pristanic acid is further converted by a synthase reaction to pristanoyl-CoA, and
is used for β- oxidation. Phytanoyl-CoA and pristanoyl-CoA, thus, are the substrates for α-
and β- oxidations, respectively [173].
Another bottle neck in the degradation of PA is the presence of an R-form [177]. The
enzymes responsible for β- oxidation are stereospecific [178], and as such, an α-
methylacyl-CoA racemase (AMCAR) is required to convert 2R-pristanoyl-CoA to its 2S
isomer before it can be oxidized [93]; see Figure 11. At first, two cycles of β-oxidation, give
rise to 2, 6, 10 trimethylundecanoyl-CoA, which is R-configured. A racemase action
converts this product into its S-isomer, and a third β-oxidation is performed to generate 4
,8 dimethylnonanoyl CoA and short-chain acyl-CoAs [179]. These products of peroxisomal
β-oxidation are either hydrolyzed to acids (route 1) or esterified to carnitine esters by the
enzymes carnitine O-octanoyltransferase (COT) and carnitine acyltransferase (CAT), and
eventually transported to the mitochondria for further β-oxidation or processing (route 2)
[159, 173]. Besides acetate, propionate has been identified as the main degradation product
of PA oxidation [173, 180].
PA can also be degraded via omega (ω)-oxidation. For more information on ω-oxidation of
PA, see the reviews by Komen et al. (2004) [181] and Wanders et al. (2011) [182].
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Figure 11: Peroxisomal α- oxidation and subsequent β-oxidation of PA (Adopted with
modifications from [173])
Phytanoyl-CoA and pristanoyl-CoA are the substrates for α- and β- oxidation, respectively.
Hydrolysis or esterification of the final peroxisomal oxidation products are transport to the
mitochondria for further processing.
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1.9.3 Milk fat composition of PA as affected by cow feed
Organic farming practices and consequently milk fat composition vary with climate and
country [183]. More so, differential feeding strategies have been shown to improve the
quality of milk [184, 185]. Grazing or green grass feeding is an obligatory part of organic
farming in Denmark but other components of feed include maize and concentrates. Being
a by-product of chlorophyll, the content of PA in milk fat ought to correlate with greed feed
intake and has been reported so in various studies [169, 186]. As such, the content of PA in
organic milk has been proposed as a marker for organic dairy products [169]. In addition,
the distribution of PA diastereomers can be used for a better authentication of organic milk
[187]. It was recently shown that the distribution of PA diastereomers changes from more
to less RRR PA, when feeding shifts from green feed (organic) to concentrates
(conventional) [165, 188]. However, very little is known of the physiological roles of the
diastereomers of PA.
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2 RESULTS AND DISCUSSION
In this section, the results regarding the use of primary porcine myotubes as a model for
skeletal muscles to study glucose and FA metabolism will be discussed. Furthermore, the
results of the effects of PA on the metabolic parameters, glucose uptake, glycogen
synthesis, and FA β-oxidation, shall be discussed in relation to glucose and FA
homeostasis. The effects of excess PA in humans shall be adressed and the impact of cow
feed on the content of PA in milk-fat will be elaborated. Finally, the metabolic implications
of the distribution of PA diastereomers in humans shall be discussed.
2.1 Primary porcine myotubes; a model of skeletal muscles, for studying
glucose and FA metabolism
The muscle is the major site for insulin-dependent glucose uptake [70, 189] and the initial
site in the generation of IR observed in T2D [189]. Therefore, the muscle represents an
ideal model to study the metabolic effects on glucose and FA homeostasis. The porcine
primary myotube model is similar to human myocytes in many ways [190], making it an
excellent choice. More so, because they are primary cells, they have not been modified in
any way other than enzymatic or physical dissociation from tissues, during purification.
Using well-established methods [191], SCs obtained from the semi-membranosus of
piglets were successfully proliferated and differentiated into multinucleated myotubes in
culture for use in this project (Figure 1, M I). As hypothesized, the porcine myotubes were
efficient for use in the analyses of glucose and FA metabolism.
In the analysis of glucose uptake, the myotubes were observed to harbor functional
insulin- mediated and non-mediated pathways, as revealed by their response to insulin
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and by the inhibiting action of cytochalasin B in both pathways (Figures 2, M I). GLUT-1
and 4 were responsible for the basal and insulin-stimulated glucose uptake, respectively,
an observation that is common in human muscles [192], but the fact that 20 % of the
glucose taken up by the myotubes could not be accounted for by neither GLUT-1 nor
GLUT-4 denotes that there might be other transporters in the pig model that aid in the
uptake of glucose. Actually, GLUT-10, 11, and 12 are, for example, known to be expressed
in mammalian skeletal muscles, and may be involved in glucose uptake, as they bind to
glucose with different affinities [193-195]. So there is a possibility that these GLUTs are
also present in pigs. It should be noted that cytochalasin B independent glucose uptake of
about the same magnitude (20 %) has been observed before [196].
GLUT-1 and 4 each activated glucose uptake in the porcine myotubes by about 4o %
(Figure 2b, M I).The GLUT-4-mediated uptake seem low when compared to a study by
Nedachi et al. (2006), where 70 % of glucose uptake was insulin-mediated [70]. This
disparity could be due to the fact that Nedachi et al used a murine cell line for the glucose
uptake analysis, and furthermore, cell lines are known to produce more proteins and
GLUT-4 than primary cells, probably because cell lines do not age or become senescent as
normal cells [197].
Another explanation of the low level of insulin-mediated uptake registered could lie in the
fact that the myotubes generated for this study were isolated from the semi-membranosus
muscles (SM) of piglets, which consists predominantly of white-type fibers. White-type
fibers are known to be low in their contents of GLUT 4 and adiponectin [198, 199], both
factors of which are necessary for the insulin-mediation of glucose uptake in the muscles.
Actually, we have seen in our lab that red-type vastus intermedius muscles (VI) show 30 %
increased insulin-mediated glucose uptake, when compared to SM (unpublished data).
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In muscles, up to 80 % of the insulin-activated glucose uptake is converted into glycogen
[200], underlining the importance of glycogen synthesis in the regulation of glucose
metabolism. In this study, the induction of glycogen synthesis in the porcine myotubes by
insulin was also effective (Figure 4b, M I), and hinted the up-regulation and activation of
GS [78]. In a previous study on insulin signaling in primary porcine myotubes, insulin
administration did not activate GS [201]. This controversial finding probably lies in
experimental methodology, rather than in the pig culture as a model, since cultures in the
latter example were exposed to supra-physiological concentrations of insulin for several
days before the analysis was performed [201]. The long-term exposure to insulin is the
likely cause of insulin-insensitivity, as hyperinsulinemia has been shown to cause IR [202].
More so, it is possible, that the chosen pigs for SC isolation was generally insulin
insensitive, especially as glycogenin which is also a rate limiting factor in glycogen
synthesis [203], was unaffected in the study [201]. We have seen in our lab that myotubes
isolated from different pigs vary in their insulin-regulated glucose uptake, with some
myotubes being completely insulin-resistant (unpublished data).
The porcine myotubes were also able to metabolize FAs. Their regulation of FA metabolism
seemed to lie in both the areas of uptake and oxidation of FAs. This is because the same
conditions that caused the generation of acetylcarnitine (C2); the end product of β-
oxidation, did not change or even reduced the degradation of palmitoylcarnitine (C16)
(Figures 4a and b, M II).
The myotubes could also be rendered resistant in both the uptake of glucose (Figure 5a, M
I) and the oxidation of FAs (Figure 3c, M II) using high concentration of glucose and
palmitate, respectively; a feature that is characteristic of human muscle cultures.
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55
2.2 The metabolic impact of PA
During glucose uptake analysis, it was shown that physiological amounts of PA enhanced
glucose uptake in the primary porcine myotubes, especially at low concentrations of
insulin (Figure 4a, MI), while further increments in the concentration of PA did not further
improve glucose uptake (Table 2, MI). This finding revealed that PA and insulin may have
similar mode of action in activating glucose uptake, as a combination of both compounds
did not have any synergistic effect on the activation of glucose uptake. In a human
intervention study, where PA-rich dairy fat was administered, there were no significant
changes in the risk markers of MS (Cholesterol, triglycerides, C-reactive protein, serum
insulin and serum glucose) [204]. Especially the observation of an unchanged serum
glucose level after the intake of PA-rich fat [204], was controversial to the results in our
project, and could be attributed to other involved factors that are present at the organism
level. As shown by the involvement of many tissues in IR [25, 32], the regulation of glucose
involves many organs, and studies of the metabolic effect of PA in an organism, as a whole,
presents an intermix of many pathways whose results can be different from those obtained
from studies in single tissue (myotubes).
It was observed that the time-dependent activation of glucose uptake by PA was biphasic,
as incubation of myotubes with PA for 4-8 h turned to increase glucose uptake, while
incubation for 12-16 h reduced glucose uptake and by 24-h incubation, there was an
increase again in glucose uptake (Figure 3a, MI). It was suggested that depletion of GLUTs
was the reason for the drop in glucose uptake, but given that the preceding rise in glucose
uptake was mild, other factors must have been involved in the biphasic effect.
PA-induced glucose uptake was significant after 60 min of incubation with glucose,
although at this time, the glucose uptake process seemed saturated (Figure 3b, MI);
B r i t a N g u m C h e P h D T h e s i s
56
glucose uptake during the first 15 min was steepest when compared to the last 15 min. This
observation showed that other factors are involved in the regulation of glucose uptake,
when PA is involved.
We showed that the glucose taken up by the myotubes in the presence of PA could not be
incorporated into glycogen, unless insulin was available (Figure 4b, MI). This observation
insinuates that the mechanism, of which PA activates glucose uptake, is different from that
which regulates glycogen synthesis. Thus, PA regulates glucose homeostasis in both an
insulin-dependent and insulin-independent manner. Our results also show that pathways,
other than glycogen synthesis are involved in the metabolism of the PA-incorporated
glucose.
When the myotubes were rendered insulin resistant by exposure to excess glucose (Figure
5b, MI), it was not possible for PA or insulin to stimulate glucose uptake or glycogen
synthesis (Figures 5c and d). One could, therefore, suggest PA to be beneficial in the
regulation of glucose in normal individuals, who generally have very low or insignificant
content of PA in their serum, or in persons that have inadequate amounts of insulin in
their serum.
During the analysis of β-oxidation in the myotubes using acylcarnitine profiling, PA
enhanced the content of C2 generated in the myotubes, as well as reduced the content of
C16, as shown by a reduced C16/C2 level (Figures 5a and b, MII). These findings denote
that PA, possibly in a PPAR-manner [133, 205, 206] improves β-oxidation by both
enhancing the degradation of C16 and increasing the content of C2.
The effect of PA on β-oxidation was not dose-dependent but longer incubation periods
with PA increased β-oxidation. During 4 h incubation of myotubes with PA, no change was
B r i t a N g u m C h e P h D T h e s i s
57
observed in the content of C16 (shown as C16/C2) and C2, but after 24 h incubation, the
content of C2 increased as the level of C16 fell and the tendency continued even when the
exposure to PA was increased to 48 h (Figure 5a and b, MII). PA has been shown to
improve FA uptake [207], which could be a reason for the unchanged level of C16, during 4
h incubation. To concretize the time-dependent effect, a lower concentration of PA (5 µM ),
which could not reduce the content of C16 or increase the level of C2 during exposure for 4
h, actually reduced and increased the levels of C16 and C2, respectively, after exposure for
48 h. These findings insinuate that PA needs time (possibly to generate more potent
metabolites [208]) to exert its effect on β-oxidation.
The fact that 5 µM PA exposed to the myotubes for 24 h caused a reduction in the content
of C16, but not an elevation of C2, suggests that PA might affects β-oxidation at different
levels and different magnitudes. Experiments with oleic acid (OLA); a saturated FA that
has been shown to have PPAR characteristics [209], revealed that it also improves β-
oxidation, at least at two levels, just like PA; at the level of uptake of FAs into the cell
and/or mitochondria, and at the level of β-oxidation of FAs. The regulation by PA, of
intermediate carnitines at the level of acyl-dehydeogenases, carnitine acyl-transferases and
MTPs cannot be excluded as a means of regulating FAO. In fact, the metabolism of PA is
known to up-regulate octanoyl-transferase [210], and possibly propionyl-transferase, given
that propionate is one of the main products of PA degradation [104, 173]. Octonoyl-
transferases have been implicated in the induction of β-oxidation of VLCFA and MCFA
[211].
Despite the observations regarding the induction of β-oxidation by PA, we could not
unequivocally conclude if it was via a PPAR-manner, since a PPAR control agonist was not
included in the study. Thus, the use of a positive control, for example a PPARα agonist
B r i t a N g u m C h e P h D T h e s i s
58
such as bezafibrates or the well-known synthetic agonist; WY-14643 [133, 205, 212], is
necessary to validate the results.
2.3 Concerns regarding excess content of total PA in humans
It is a desire to increase the total content of PA in milk fat, to achieve levels that are
physiologically advantageous. The PA content in normal human subjects that consume
ruminant products has been documented to range from 0.04-11.5 µM, and consumers of
non-ruminant products have negligible amounts of PA in their plasma, compared to
consumers of ruminant products, whose total content of PA is in the upper range [204,
213, 214]. In our studies, 10 µM PA was utilized as standard concentration as it lies within
the physiological range. More so, the viability of the myotubes were negatively affected
when the concentration of PA was over 10 µM (table 1, M I), and results from FAO
experiments with 20 µM PA where not generally better, when compared to results
achieved with 10 µM (Figure 5 b and c, M II).
2.4 PA content in milk-fat as affected by cow feed
As shown in Figure 1, M III, two naturally-occurring stereomers of PA were identified in
milk-fat, with the SRR form dominating, just as in previous findings [165]. Milk-fat
produced organically or even by conventional feeding has been shown to have higher
contents of PA than in our study [187, 188]. This discrepancy in the content of PA lies in
different farming practices between farms or regions/countries.
B r i t a N g u m C h e P h D T h e s i s
59
Surprisingly, we found that an increase in the total content of PA was attributed to feed
concentrates, while the reverse was attributed to the content of pasture in feed (Table 2, M
III). These correlations were, however not very strong and could be attributed to random
variation. Nevertheless, our finding could actually explain the difference in PA contents
between the grazing seasons. The total content of PA was higher in September than in
May, and this is probably because the use of concentrates was generally augmented in
September to compromise for the weather-mediated low dry matter intake (DMI) from
grazing in September, and not because of the content of legumes that constituted a larger
part of pastures in September. Nevertheless, it could be that the availability of light during
the May season (there is generally more light in the May than September season), also
affected the results. The degradation of chlorophyll in leaves have been shown to be
enhanced by photo-oxidation [215]. In this case, the production of phytol should be
enhanced and, thereby generating more PA during the May season. But given our findings,
it is obvious that other factors (e.g. the developmental state of the plants) affect the
production and content of PA in milk-fat more.
We suggested that the use of grass pellets in concentrates was the cause for the elevated
content of total PA, since grass feed has been shown to generate higher contents of PA
[186]. Actually, dried grass has been shown to increase the degradation of chlorophyll in
sheep rumen [216]. As PA can only be produced from free phytol, chlorophyll degradation,
therefore, will increase the production of PA.
The inverse correlation of pasture to the total content of PA observed in our studies
contradicts previous conclusions that green feeding increase the content of PA [169, 217].
In our experiments, pasture was dominated mostly by legumes (ryegrass and white clover).
Howard et al. (1993) found that legumes like soya beans present a “stay green”
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60
phenomenon, whereby chlorophyll degradation is resisted [218]; maybe this phenotype is
common for the legumes in the pasture used in our study, and would thereby explain the
decrease in the total content of PA recorded. Nevertheless, as we found no correlation
between the total content of PA and legumes, it is obvious that other unknown factors are
involved in the production of PA. A study conducted by Lee et al. (2006) showed that the
total content of PA in duodenal flow was reduced in line with a reduction in
biohydrogenation [186]. Although this trend was affected by the intake of red clover, it is
likely that, partly the influence of legumes on biohydrogenation is causing the reduction in
the total content of PA after pasture intake, observed in our study.
Many factors other than proteolytic activities in the pasture might alter ruminal micro
flora [219], and, thus, responsible for driving the mechanisms behind our observations of
altered PA content with feed. The rate limiting step in the catabolism of chlorophyll, which
generates free phytol, is controlled by the chlorophyllase and has been showed to be post-
translationally regulated [157], possibly by a well of factors, some of which can originate
from the rumen of the cow. In that case, the feed can have an influence on chlorophyllase.
Phytol exists in two forms (E- and Z- form) and the degradation and production of each
form from chlorophyll depend on the bacteria community [161, 220], whose population
and action can be affected differently by the feed of the cow. Moreover, the enzymes
involved in the degradation of phytol are stereospecific, favoring the degradation of E-
phytol, and these enzymes can be altered by the feed [159].
The values of the total content of PA obtained in our study do not necessarily indicate the
lack or limited use of green feed in organic farming, but merely indicate that the type of
(green) feed can alter the acquired content of total PA in milk-fat. It is apparent, therefore,
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61
to suggest that it is the feed composition that in many levels and ways controls the total
content of PA.
To support this suggestion, we also showed that although pasture did not affect the total
content of PA, the increasing content of legumes in the pasture caused an elevation in the
content of RRR PA (Table 2, M III). Schröder et al. (2011 and 2012) also reported that
organic feeding, which is dominated by green feed (grass silage) or conventional feeding
rich in concentrates, increased the content of SRR PA in milk-fat, while organic feeding
dominated by hay feeding increased the content of RRR PA instead [187, 188]. Likely
reasons for the altered content of PA diastereomers are similar to some of those that
caused an alteration in the total content of PA; namely, the altered distribution or action of
ruminal micro flora bacteria in response to feed, and also the stereospecificity of
degradation enzymes. As mentioned earlier, β-oxidation enzymes prefer SRR-pristanic
acid (generated from SRR PA) as substrate, and in that case, one would expect to have
different contents of the diastereomers in milk. More so, effects on AMCAR (enzyme
necessary to convert RRR PA to SRR PA) by feed may be possible.
2.5 Concerns regarding the distribution of PA diastereomers in
humans
The notion to elevate the total PA content of milk fat was borne with little knowledge of the
distribution of the individual stereomers of PA and what impact the diastereomers might
have on the physiology. Studies carried out by Tsai et al. (1973) showed that SRRR PA
instead of RRR PA was preferred for oxidation [170], but this conclusion was drawn from
experiments with very high concentrations of PA, like those found in a diseased state, and
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62
revealed nothing about the PPAR-effects of PA. Nevertheless, Heim et al. (2002) later
showed that both forms of PA affected PPARs equally [124], but this study was performed
just in one tissue; the liver. Results from other tissues or organs would give more
convincing observations and information on the effects of PA diastereomers.
The knowledge that β-oxidation of FAs is stereospecific [178] with preference towards the
SRR form generates interests in the diastereomatic distribution of PA in milk fat. Because
of a methyl side chain on position C3, the metabolism of PA occurs through α- oxidation in
the peroxisome, resulting in a more potent PPAR-inducer; pristanic acid [208]. Pristanic
acid existing in the RRR-form has to be converted by AMCAR to the SRR form, for it to be
β-oxidized. These bottle-necks in the metabolism of PA create a scenario with regards to
what feeding strategy to embark on. On the one hand, feed composition that will give a
higher ratio of SRR/RRR PA could be thought desirable, as its metabolism would be
preferably faster and less dependent on AMCAR. On the other hand, it is hard to conclude
that milk-fat with higher RRR/SRR PA ratio is undesirable; one can speculate that a slower
degradation of RRR PA may cause an increase in the level of pristanic acid, and since
pristanic acid is a more potent PPARα agonist than PA, the expression of PPARα would be
up-regulated. However, it is important that the elevated level of PA or pristanic acid is
within a physiological range.
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3 CONCLUDING REMARKS
As illustrated in Figure 12, we show in this study that manipulating the feeding strategy of
the cow can alter the total content and distribution of PA diastereomers in the milk-fat of
the cow. More so, we could show that the exposure of a physiologically relevant
concentrations of a mixture of PA isomers induced glucose uptake and β-oxidation in
primary porcine myotubes, which we established as an efficient skeletal muscle model.
However, the mechanisms behind the control of glucose and FA metabolism by PA remain
to be investigated.
Figure 12: An illustration of the effects of cow feed on the total content of PA and its
diastereomers, and the metabolic impact of PA on skeletal muscles
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64
4 PERSPECTIVES
During glucose uptake analysis, we found that PA at physiological concentrations
enhanced glucose uptake but not glycogen synthesis. It would, thus, be relevant to
investigate the fate of the glucose after uptake and whether other metabolic pathways of
glucose, than glycogen synthesis are affected by PA. While PA, in the presence of insulin,
generated a synergistic effect on the activation of glycogen synthesis, no such effect was
observed during glucose uptake. These observations insinuate that more than one
mechanism is involved in the PA-mediated metabolism of glucose, and needs to be
analyzed further.
PA was observed to enhance the degradation of C16 and increase the production of C2.
However, a positive PPARα control, such as bezafibrates, needs to be investigated in
parallel with PA, before we can conclude on what mechanism is responsible for this effect.
More so, the effect of PA on intermediate acylcarnitines is relevant to get an overview of
the general impact of PA on FA metabolism.
In our studies, synthetic PA was used. Given that this product is a mixture of up to eight
isomers of unknown distribution, it is hard to tell whether our in vitro finding concerning
the metabolic impact of PA could have been better, if a pure PA product consisting only of
the two naturally known isomers had been used. Thus, studies with the purified naturally
occurring forms of PA are of interest. The finding that RRR PA can be enhanced by cow
feed makes it even more relevant to elucidate the metabolic effects of this form. Also,
analysis of other tissues than muscles would give more information about the distribution
and effects of PA diastereomers.
B r i t a N g u m C h e P h D T h e s i s
65
Gene expression experiments and protein determination of specific markers of glucose
uptake and FAO are necessary to fully concretize the findings in our studies. In this regard,
human muscle cell lines could be used as a supplement to the primary porcine model,
since cell lines generate enough proteins for biochemical and histological studies. More so,
the human muscles express uncoupling proteins that regulate thermogenesis, and this
process has been shown to be regulated by PA. Even more interesting is the fact that the
use of cell line would exclude constrains of dealing with variation between pigs, although
this factor is a natural aspect that needs to be kept in mind when dealing with cell lines.
Which of the natural forms of PA, that influence this metabolic process could be of
relevance.
In our analysis with Jersey and Holstein cows, we could not link breed to the total content
of PA or distribution of its diastereomers. As the composition of feed intake varied between
breeds, one can speculate whether the conclusion would have been the same, had it been
the same variation in feed composition applied to both breeds. Such an experiment could
be of relevance.
Drawing from the observations in this project, the desired content of PA and distribution
of its diastereomers in milk-fat can be achieved by manipulating the composition of the
cow feed. Therefore, it will be interesting to analyze the impact of the legumes and other
feed components commonly used in Denmark, on the total content PA and its
diastereomatic distribution. Analysis from the level of chlorophyll degradation, through
the breakdown of the two forms of phytol, micro flora involvement, and ruminal
production and degradation of the two forms of PA would be relevant.
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6. APPENDIX
6.1 Manuscript I; MI
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Phytanic acid stimulates glucose uptake in a model of skeletal
muscles, the primary porcine myotubes
Brita N. Che1, Niels Oksbjerg1, Lars I. Hellgren2, Jacob H. Nielsen3, Jette F. Young1*
1: Department of Food Science, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark
2: Department of System Biology, Technical University of Denmark, 2800 Kgs. Lyngby,