Talk:Fatty acid oxidation

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O2k-Network discussion forum: fatty acids used in permeabilized fibre assays (2015-10-20)


 Experience with FAO in PFIFatty acid usedMammal and modelTissue and cellCoupling statesPathways
Baekkerud 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitine + PalmitoylcarnitineMouseF
Bouitbir 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineHeart
Skeletal muscle
F
Cannon 2015 Fatty Acid Oxidation O2k-Network Discussion Forum
  • Saponin permeabilized skeletal muscle fiber bundles
  • Protocol:
  1. 0.5mM Malate
  2. 50 µM palmitoyl-CoA + 2mM carnitine
  3. 5mM ADP
  4. From here on out, various combinations for titration protocol
  • Coupling states:
  1. LEAK_M+Palmitoylcarnitine
  2. P_M+Palmitoylcarnitine
  3. P_M+Palmitoylcarnitine+S
  4. E_O+CCCP titrations
  5. E_S+Rot
  6. ROX_AntimycinA
PalmitoylcarnitineHuman
Mouse
Skeletal muscleLEAK
OXPHOS
ET
F
N
S
NS
ROX
Chavanelle 2015 Fatty Acid Oxidation O2k-Network Discussion ForumFatty oxidation in permeabilised db/db mice muscle fibres, in Mir05 + Cr +37°C. We found that ADP-stimulated respiration in presence of 0,02 mM of palmitoyl-carnitine (OXPHOS) is very close to the non-ADP-stimulated respiration (LEAK state) when working with these conditions. (Values around 14 to 20 (pmol/s/mg w.t.) for LEAK state and 15 to 24 (pmol/s/mg w.t.) for fatty acid triggered-OXPHOS). These data suggest either a very poor ability for this animal model to use palmitoylcarnitine or a very high LEAK state (or both!)

Protocol

  1. 2,2 mL mir05+Cr was added in the chambers and oxygen was added to the medium up to 400 – 450 nmol/mL
  2. Chambers were closed
  3. 10µM Amplex Red was added to the chambers
  4. 2 U HRP was added
  5. 10 U SOD was added
  6. 20µM Palmitoyl-Carnitine was added
  7. 2 mM Malate was added
  8. 1 to 3 mg wet permeabilised muscle fibers was added
  9. LEAK state was recorded
  10. Mg+ was added
  11. 2,5 mM ADP was added
  12. Pal-C stimulated OXPHOS was recorded
The tissue used was skeletal muscle (Gastrocnemius)
PalmitoylcarnitineMouseSkeletal muscleLEAK
OXPHOS
F
N
Chicco 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have used palmitoylcarnitine for most of our studies, but have found it to behave inconsistently in the past, and differently than OctCrn when the two have been compared. So, we have used both, but would be best represented by Palmitoylcarnitine.PalmitoylcarnitineF
Chou 2015 Fatty Acid Oxidation O2k-Network Discussion ForumProtocol: final concentration of digitonin in chamber is 10μg/ml cell number in chamber is 2 millions cell/ml, cell type PBMC, Malate (2mM), Palmitoyl-DLcarnitine-HCl (20μM), ADP (2.5mM), pyruvate (5mM), glutamate (10mM), succinate (10mM), rotenone (0.1μM), malonic acid(5mM), myxothiazol (0.5μM), antimycin A (2.5μM), TMPD (0.5mM), azide (100mM)Palmitoylcarnitine
TW
Blood cells
Lymphocyte
Platelet
OXPHOS
ET
F
N
S
NS
ROX
Ciapaite 2015 Fatty Acid Oxidation O2k-Network Discussion ForumProtocol: I use either palmitoyl-L-carnitine plus malate (25 µM + 2.5 mM) or palmitoyl-CoA + L-carnitine + malate (25 µM + 2 mM + 2.5 mM) as substrates. Respiratory control index is usually around 5-6 for healthy controls.Palmitoylcarnitine
Palmitoyl-CoA + L-carnitine + Malate
Human
Rat
Skeletal muscleLEAK
OXPHOS
ET
F
Coen 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use Palmitylcarnitine as a substrate in assays of permeabilized fiber bundle respiration. The substrate works well for us and in combination with Pal, Mal, Glut, and Succ we see higher levels of state 3 respiration than without it.PalmitoylcarnitineOXPHOSF
N
NS
Darveau 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI have conducted one study examining bumblebee flight muscle permeabilized fibers and used.PalmitoylcarnitineHexapodsSkeletal muscleF
Egelandsdal 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have examined for how long time post mortem the fatty acid oxidation prevails in beef muscles.OctanoylcarnitineBovinesSkeletal muscleF
Essop 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Fontes-Oliveira 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitine + MalateF
Goodpaster 2015 Fatty Acid Oxidation O2k-Network Discussion ForumExcellent. Lower overall O2 consumption compared to carbohydrate-supported respirationPalmitoylcarnitineF
Gorr 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWhat´s wrong with using simple C16 saturated or C18 monounsaturated fatty acids like palmitate or oleate, respectively, for analysing beta-oxidation rates??F
Gottschalk 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Hansson Fatty Acid Oxidation O2k-Network Discussion ForumWe have only performed limited pilot studies with fatty acids in permeabilized heart fibers. In general the respiration by both octanoylcarnitine and palmitylcarnitine have been quite low. We have not optimized the settings so we do not have much valuable experience to share.Octanoylcarnitine
Palmitoylcarnitine
HeartF
Hickey 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use Palmitoyl Carnitine, I recall we made it up with fatty acid free BSA at 5:1 (PC:BSA). Worked well with fish and rat heart mitochondria, and permeabilised fibres. Protocol: Palmitoylcarnitine we made it with fatty acid free BSA at 5:1 (PC:BSA)PalmitoylcarnitineRat
Fishes
HeartF
Jelenik 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe standardly measure FA-linked respiration in the muscle - we used Octanoyl-C as well as Palmitoyl-C, however, the latter one gives poor response. Thus we use octanoyl.OctanoylcarnitineF
Kane 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Lemieux 2015 Fatty Acid Oxidation O2k-Network Discussion ForumHeart and skeletal muscle. Permeabilized animals (Dugesia tigrina worm) Other substrates: Acetylcarnitine + Malate, Palmitoyl-CoA+Malate+CarnitineOctanoylcarnitine + Malate
Palmitoylcarnitine + Malate
Heart
Skeletal muscle
F
Lesnefsky 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use the method pioneered by Dr. Chuck Hoppel in Cleveland. We use palmitoylcarnitine. To assess role of CPT-1; one can use palmitoyl-CoA + carnitinePalmitoylcarnitine
Palmitoyl-CoA + Carnitine
F
Ljubkovic 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI am sending you a graph with some mitochondrial respiration measurements obtained from the left ventricular human tissue of patients undergoing CABG surgery. As can be seen from the graph, we used FFA substrate to evoke the State 2 and State 3 respiration, and then followed with other substrates and an uncoupler. FFA substrate was palmitoyl-carnitine (40 µM concentration). We performed quite a few of experiments using palmitoyl-carnitine, also in the range of concentrations from 1 – 40 µM. We usually see a gradual increase in State 3 respiration with concentration increments (1, 2.5, 5, 10, 15, 40 µM). However, the respiration is always lower then when pyruvate is used (although in much higher concentration, from 2 mM up). All of the observations come from the human left ventricular tissue, that we almost exclusively use in past couple of years for our experiments on mitochondria (we also measured activity of some enzymes and protein expression).PalmitoylcarnitineHumanHeartOXPHOS
ET
F
N
NS
Madlala 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have assessed beta oxidation in permeabilised skeletal muscle fibres. We initially used palmitoylcarnitine in our analysis but we realised that using this substrate for assessing beta oxidation in skeletal muscle of rat does not work (no response in respiration). This is also documented in literature; absence of LCAD in rat skeletal muscle so we decided to use octanoylcarnitine instead and it works well. Afterwards the muscle is responsive to ADP and uncoupler titrations. However, we use this in combination with malate.Octanoylcarnitine + Malate
Palmitoylcarnitine
RatSkeletal muscleOXPHOSF
Makrecka 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have examined fatty acid oxidation in permeabilized muscle fibres and cardiac fibres and in isolated cardiac mitochondria of rats and mice. Since the area of our research is the regulation of energy metabolism, the determination of fatty acid oxidation is one of the typical measurements. Which substrate we use, is dependent on the aim (include CPT I (rate limiting step) or not). In addition, presence of BSA in media and used carnitine concentration are always under discussions (physiological or not physiological).Palmitoylcarnitine + MalateMouse
Rat
Heart
Skeletal muscle
F
McCurdy 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have had good success using the Octanoylcarnitine at the recommended dose of 0.2mM in SUIT protocols with PMG and with just GM. Respiration increases with addition of ADP and subsequent substrate additions. We have also used palmitoylcarnitine at a conc of 0.025 -0.05mM with mixed success. Often there is no or little stable increase in ADP stimulated respiration and requires addition of pyr and/or Glut. We primarily test nonhuman primate (japanses macaque) permeabilized muscle fibers.Octanoylcarnitine + PalmitoylcarnitineOther mammalsSkeletal muscleOXPHOSF
N
Miller 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitineF
Moellering 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use submaximal concentrations 40µM of palmitoylcarnitine with our permeabilized muscle fiber preps.PalmitoylcarnitineSkeletal muscleF
Neviere 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineHuman
Mouse
F
Ojuka 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have used Octanoylcarnitine + malate as fatty acid substrates (at the concentration stipulated in BIOBLAST) with much success. However, Palmitylcarnitine + malate was not responsive to ADP or CCCP.Octanoylcarnitine + MalateOXPHOS
ET
F
Ost 2015 Fatty Acid Oxidation O2k-Network Discussion ForumRight now we´re using octanoyl carnitine (OC) when examining mouse skeletal muscle permeabilized fiber respiration. We ususally start with Malate (2mM) and OC (0.2mM), then add ADP (5mM) in order to assess fatty acid oxidative capacity. For Soleus muscle fibers we got an average respiration of 60 pmol/(s*mg fiber ww).Octanoylcarnitine
Malate
ADP
MouseSkeletal muscleF
Pasdois 2015 Fatty Acid Oxidation O2k-Network Discussion ForumProtocol: Couple palmitoylcarnitine (10µM) + malate (1mM) on isolated mitochondria and permeabilized fibers. In such case the buffer is always supplemented with 2mg/ml of BSA.Palmitoylcarnitine + MalateF
Ramsay 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI recommend that everyone uses palmitoyl-Cn (with BSA of course) for the most physiological measurement. I actually used decanoyl-CoA when I wanted a more soluble substrate.Palmitoyl-Cn
Decanoyl-CoA
F
Roden 2015 Fatty Acid Oxidation O2k-Network Discussion ForumGreat experienceOctanoylcarnitine + PalmitoylcarnitineHuman
Mouse
Skeletal muscleF
Salin 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWorked a bit with palmitoylcarnitine on homogenate of white muscle of fish. Do not need to be permeabilized chemically as mechanical permeabilization allow to obtain permeabilized cell (ADP-responsiveness the same). The respiration rate was much lower than the pyruvate / malate / succinate substrate, so I stopped to use the lipid substrates.PalmitoylcarnitineFishesSkeletal muscleF
Schauer 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe are successfully measuring FA oxidation in permeabilized fibers. We have switched to Octanoylcarnitine (A) because we were seeing evidence of toxicity with palmitoylcarnitine. This evidence was that we were seeing final uncoupled respiration to be lower than state 3 respiration in samples with PC. This issue resolved when we switched to OC.OctanoylcarnitineOXPHOS
ET
F
Senyilmaz 2015 Fatty Acid Oxidation O2k-Network Discussion ForumInverted Drosophila larvae which are permeabilized by digitonin. Their body walls are more or less made of muscles.Palmitoyl-CoA + CarnitineDrosophilaSkeletal muscleF
Smith 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI have extensively tested FAO in PMFBs using the Palmitoylcarnitine, Palmitoyl-CoA, and L-Carnitine. Protocol: For maximal respiration we make sequential additions of 18 µM PC, 20 µM PCoA, and 5 mM Carnitine. Addition of ADP induces a peak rate of O2 consumption that is not increased with FCCP addition.Palmitoylcarnitine
Palmitoyl-CoA
and L-Carnitine
OXPHOS
ET
F
Sparks 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe routinely (up to 4 human and mouse studies) measure respiration supported by glycolytic and fatty acid substrates using SUIT protocols in permeabilized muscle fibers. We always use palmitoylcarnitine. In the past, in Dr. Schrauwen’s lab, we used octanoate to titrate for fatty acid induced uncoupling.Palmitoylcarnitine
Octanoate
Human
Mouse
Skeletal muscleF
Van Schaardenburgh 2015 Fatty Acid Oxidation O2k-Network Discussion ForumIn my studies on patients with intermittent claudication we used Octanoylcarnitine in the SUIT protocol described by Pesta and you.OctanoylcarnitineF
Veksler 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe studied fatty acid oxidation in permeabilised cardiac and skeletal muscle fibers for 15 years. We use palmitoylcarnitine, palmitoyl-CoA or octanoatePalmitoylcarnitine
Palmitoyl-CoA
Octanoate
Heart
Skeletal muscle
F
Votion 2015 Fatty Acid Oxidation O2k-Network Discussion ForumVery limited (only preliminary tests on mice) but satisfying. I have planned new experiments on equine muscle tissue in 2016.Octanoylcarnitine
Palmitoylcarnitine
Octanoate
Acetylcarnitine
Palmitoyl CoA (+ Carnitine)
MouseF
West 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitineF
Wohlwend 2015 Fatty Acid Oxidation O2k-Network Discussion ForumFatty acid oxidation in permeabilized muscle fibers has worked well for us in both human and rat studies. Typically we use Octanoylcarnitine and Malate for skeletal muscle and a combination of Octanoylcarnitine plus Palmitylcarnitine and Malate for human heart tissue. These protocols have given us a good insight into Beta oxidation.Octanoylcarnitine + Malate
Octanoylcarnitine + Palmitoylcarnitine + Malate (for heart)
Human
Rat
Skeletal muscleF
Wright 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe do include octanoylcarnitine in the initial titration of substrates for our SUIT protocols typically, but do not measure its individual contribution to OXPHOS.OctanoylcarnitineF
Wuest 2015 Fatty Acid Oxidation O2k-Network Discussion ForumCompared with just glucose oxidation, the max OXPHOS/ET capacity was slightly (but significantly) lower with PC & Oct. Protocol: PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA. The concentrations were the same as in the blue book.Palmitoylcarnitine + OctanoylcarnitineRatHeartOXPHOS
ET
F
N
S
NS
ROX

O2k-Network discussion forum: fatty acids all data (2015-10-20)

 Experience with FAO in PFIFatty acid usedMammal and modelTissue and cellCoupling statesPathways
Ali 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Baekkerud 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitine + PalmitoylcarnitineMouseF
Bouitbir 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineHeart
Skeletal muscle
F
Cabrera-Fuentes 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Cannon 2015 Fatty Acid Oxidation O2k-Network Discussion Forum
  • Saponin permeabilized skeletal muscle fiber bundles
  • Protocol:
  1. 0.5mM Malate
  2. 50 µM palmitoyl-CoA + 2mM carnitine
  3. 5mM ADP
  4. From here on out, various combinations for titration protocol
  • Coupling states:
  1. LEAK_M+Palmitoylcarnitine
  2. P_M+Palmitoylcarnitine
  3. P_M+Palmitoylcarnitine+S
  4. E_O+CCCP titrations
  5. E_S+Rot
  6. ROX_AntimycinA
PalmitoylcarnitineHuman
Mouse
Skeletal muscleLEAK
OXPHOS
ET
F
N
S
NS
ROX
Chavanelle 2015 Fatty Acid Oxidation O2k-Network Discussion ForumFatty oxidation in permeabilised db/db mice muscle fibres, in Mir05 + Cr +37°C. We found that ADP-stimulated respiration in presence of 0,02 mM of palmitoyl-carnitine (OXPHOS) is very close to the non-ADP-stimulated respiration (LEAK state) when working with these conditions. (Values around 14 to 20 (pmol/s/mg w.t.) for LEAK state and 15 to 24 (pmol/s/mg w.t.) for fatty acid triggered-OXPHOS). These data suggest either a very poor ability for this animal model to use palmitoylcarnitine or a very high LEAK state (or both!)

Protocol

  1. 2,2 mL mir05+Cr was added in the chambers and oxygen was added to the medium up to 400 – 450 nmol/mL
  2. Chambers were closed
  3. 10µM Amplex Red was added to the chambers
  4. 2 U HRP was added
  5. 10 U SOD was added
  6. 20µM Palmitoyl-Carnitine was added
  7. 2 mM Malate was added
  8. 1 to 3 mg wet permeabilised muscle fibers was added
  9. LEAK state was recorded
  10. Mg+ was added
  11. 2,5 mM ADP was added
  12. Pal-C stimulated OXPHOS was recorded
The tissue used was skeletal muscle (Gastrocnemius)
PalmitoylcarnitineMouseSkeletal muscleLEAK
OXPHOS
F
N
Chicco 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have used palmitoylcarnitine for most of our studies, but have found it to behave inconsistently in the past, and differently than OctCrn when the two have been compared. So, we have used both, but would be best represented by Palmitoylcarnitine.PalmitoylcarnitineF
Chou 2015 Fatty Acid Oxidation O2k-Network Discussion ForumProtocol: final concentration of digitonin in chamber is 10μg/ml cell number in chamber is 2 millions cell/ml, cell type PBMC, Malate (2mM), Palmitoyl-DLcarnitine-HCl (20μM), ADP (2.5mM), pyruvate (5mM), glutamate (10mM), succinate (10mM), rotenone (0.1μM), malonic acid(5mM), myxothiazol (0.5μM), antimycin A (2.5μM), TMPD (0.5mM), azide (100mM)Palmitoylcarnitine
TW
Blood cells
Lymphocyte
Platelet
OXPHOS
ET
F
N
S
NS
ROX
Ciapaite 2015 Fatty Acid Oxidation O2k-Network Discussion ForumProtocol: I use either palmitoyl-L-carnitine plus malate (25 µM + 2.5 mM) or palmitoyl-CoA + L-carnitine + malate (25 µM + 2 mM + 2.5 mM) as substrates. Respiratory control index is usually around 5-6 for healthy controls.Palmitoylcarnitine
Palmitoyl-CoA + L-carnitine + Malate
Human
Rat
Skeletal muscleLEAK
OXPHOS
ET
F
Coen 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use Palmitylcarnitine as a substrate in assays of permeabilized fiber bundle respiration. The substrate works well for us and in combination with Pal, Mal, Glut, and Succ we see higher levels of state 3 respiration than without it.PalmitoylcarnitineOXPHOSF
N
NS
Cortassa 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoyl CoA (mitochondria) and Palmitate (conjugated with BSA) in cardiomyocytes.Palmitoyl CoA
Palmitate
Skeletal muscleF
Darveau 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI have conducted one study examining bumblebee flight muscle permeabilized fibers and used.PalmitoylcarnitineHexapodsSkeletal muscleF
Egelandsdal 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have examined for how long time post mortem the fatty acid oxidation prevails in beef muscles.OctanoylcarnitineBovinesSkeletal muscleF
Essop 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Filippi 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitine
Oleate
LiverF
Fontes-Oliveira 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitine + MalateF
Goetzman 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineLiverF
Goodpaster 2015 Fatty Acid Oxidation O2k-Network Discussion ForumExcellent. Lower overall O2 consumption compared to carbohydrate-supported respirationPalmitoylcarnitineF
Gorr 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWhat´s wrong with using simple C16 saturated or C18 monounsaturated fatty acids like palmitate or oleate, respectively, for analysing beta-oxidation rates??F
Gottlieb 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe used palmitate added to culture media overnight in differentiated C2C12 myotubes.PalmitateMouseSkeletal muscleF
Gottschalk 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Hagland 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitic acidF
Hansson Fatty Acid Oxidation O2k-Network Discussion ForumWe have only performed limited pilot studies with fatty acids in permeabilized heart fibers. In general the respiration by both octanoylcarnitine and palmitylcarnitine have been quite low. We have not optimized the settings so we do not have much valuable experience to share.Octanoylcarnitine
Palmitoylcarnitine
HeartF
Hickey 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use Palmitoyl Carnitine, I recall we made it up with fatty acid free BSA at 5:1 (PC:BSA). Worked well with fish and rat heart mitochondria, and permeabilised fibres. Protocol: Palmitoylcarnitine we made it with fatty acid free BSA at 5:1 (PC:BSA)PalmitoylcarnitineRat
Fishes
HeartF
Jelenik 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe standardly measure FA-linked respiration in the muscle - we used Octanoyl-C as well as Palmitoyl-C, however, the latter one gives poor response. Thus we use octanoyl.OctanoylcarnitineF
Kaambre 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineHumanGenitalF
Kane 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Kollberg 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Krishnan 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineLiverF
Leloup 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitateNervous system
Lemieux 2015 Fatty Acid Oxidation O2k-Network Discussion ForumHeart and skeletal muscle. Permeabilized animals (Dugesia tigrina worm) Other substrates: Acetylcarnitine + Malate, Palmitoyl-CoA+Malate+CarnitineOctanoylcarnitine + Malate
Palmitoylcarnitine + Malate
Heart
Skeletal muscle
F
Lesnefsky 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use the method pioneered by Dr. Chuck Hoppel in Cleveland. We use palmitoylcarnitine. To assess role of CPT-1; one can use palmitoyl-CoA + carnitinePalmitoylcarnitine
Palmitoyl-CoA + Carnitine
F
Ljubkovic 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI am sending you a graph with some mitochondrial respiration measurements obtained from the left ventricular human tissue of patients undergoing CABG surgery. As can be seen from the graph, we used FFA substrate to evoke the State 2 and State 3 respiration, and then followed with other substrates and an uncoupler. FFA substrate was palmitoyl-carnitine (40 µM concentration). We performed quite a few of experiments using palmitoyl-carnitine, also in the range of concentrations from 1 – 40 µM. We usually see a gradual increase in State 3 respiration with concentration increments (1, 2.5, 5, 10, 15, 40 µM). However, the respiration is always lower then when pyruvate is used (although in much higher concentration, from 2 mM up). All of the observations come from the human left ventricular tissue, that we almost exclusively use in past couple of years for our experiments on mitochondria (we also measured activity of some enzymes and protein expression).PalmitoylcarnitineHumanHeartOXPHOS
ET
F
N
NS
Lund 2015 Fatty Acid Oxidation O2k-Network Discussion ForumCarnitine + Palmitoyl CoAF
Madlala 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have assessed beta oxidation in permeabilised skeletal muscle fibres. We initially used palmitoylcarnitine in our analysis but we realised that using this substrate for assessing beta oxidation in skeletal muscle of rat does not work (no response in respiration). This is also documented in literature; absence of LCAD in rat skeletal muscle so we decided to use octanoylcarnitine instead and it works well. Afterwards the muscle is responsive to ADP and uncoupler titrations. However, we use this in combination with malate.Octanoylcarnitine + Malate
Palmitoylcarnitine
RatSkeletal muscleOXPHOSF
Mahdaviani 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineFatF
Makrecka 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have examined fatty acid oxidation in permeabilized muscle fibres and cardiac fibres and in isolated cardiac mitochondria of rats and mice. Since the area of our research is the regulation of energy metabolism, the determination of fatty acid oxidation is one of the typical measurements. Which substrate we use, is dependent on the aim (include CPT I (rate limiting step) or not). In addition, presence of BSA in media and used carnitine concentration are always under discussions (physiological or not physiological).Palmitoylcarnitine + MalateMouse
Rat
Heart
Skeletal muscle
F
McCurdy 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have had good success using the Octanoylcarnitine at the recommended dose of 0.2mM in SUIT protocols with PMG and with just GM. Respiration increases with addition of ADP and subsequent substrate additions. We have also used palmitoylcarnitine at a conc of 0.025 -0.05mM with mixed success. Often there is no or little stable increase in ADP stimulated respiration and requires addition of pyr and/or Glut. We primarily test nonhuman primate (japanses macaque) permeabilized muscle fibers.Octanoylcarnitine + PalmitoylcarnitineOther mammalsSkeletal muscleOXPHOSF
N
Miller 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitineF
Moellering 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe use submaximal concentrations 40µM of palmitoylcarnitine with our permeabilized muscle fiber preps.PalmitoylcarnitineSkeletal muscleF
Neviere 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineHuman
Mouse
F
Ojuka 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe have used Octanoylcarnitine + malate as fatty acid substrates (at the concentration stipulated in BIOBLAST) with much success. However, Palmitylcarnitine + malate was not responsive to ADP or CCCP.Octanoylcarnitine + MalateOXPHOS
ET
F
Ost 2015 Fatty Acid Oxidation O2k-Network Discussion ForumRight now we´re using octanoyl carnitine (OC) when examining mouse skeletal muscle permeabilized fiber respiration. We ususally start with Malate (2mM) and OC (0.2mM), then add ADP (5mM) in order to assess fatty acid oxidative capacity. For Soleus muscle fibers we got an average respiration of 60 pmol/(s*mg fiber ww).Octanoylcarnitine
Malate
ADP
MouseSkeletal muscleF
Palm 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitineF
Pasdois 2015 Fatty Acid Oxidation O2k-Network Discussion ForumProtocol: Couple palmitoylcarnitine (10µM) + malate (1mM) on isolated mitochondria and permeabilized fibers. In such case the buffer is always supplemented with 2mg/ml of BSA.Palmitoylcarnitine + MalateF
Ramsay 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI recommend that everyone uses palmitoyl-Cn (with BSA of course) for the most physiological measurement. I actually used decanoyl-CoA when I wanted a more soluble substrate.Palmitoyl-Cn
Decanoyl-CoA
F
Robidoux 2015 Fatty Acid Oxidation O2k-Network Discussion ForumProtocol: Palmitate, Stearate, Oleate and Linoleate in intact cells. We use various BSA-fatty acid combinations that result in free fatty acid levels that are in the 2 to 12 nM range.Palmitate
Stearate
Oleate
Linoleate
FatF
Roden 2015 Fatty Acid Oxidation O2k-Network Discussion ForumGreat experienceOctanoylcarnitine + PalmitoylcarnitineHuman
Mouse
Skeletal muscleF
Salin 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWorked a bit with palmitoylcarnitine on homogenate of white muscle of fish. Do not need to be permeabilized chemically as mechanical permeabilization allow to obtain permeabilized cell (ADP-responsiveness the same). The respiration rate was much lower than the pyruvate / malate / succinate substrate, so I stopped to use the lipid substrates.PalmitoylcarnitineFishesSkeletal muscleF
Schauer 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe are successfully measuring FA oxidation in permeabilized fibers. We have switched to Octanoylcarnitine (A) because we were seeing evidence of toxicity with palmitoylcarnitine. This evidence was that we were seeing final uncoupled respiration to be lower than state 3 respiration in samples with PC. This issue resolved when we switched to OC.OctanoylcarnitineOXPHOS
ET
F
Schoettl 2015 Fatty Acid Oxidation O2k-Network Discussion ForumBeta oxidation in adipocytes (white and brown), HEK293 cells, isolated mitochondria from liver, adipocytes, brain, etc.PalmitoylcarnitineNervous system
Liver
Fat
HEK
F
Senyilmaz 2015 Fatty Acid Oxidation O2k-Network Discussion ForumInverted Drosophila larvae which are permeabilized by digitonin. Their body walls are more or less made of muscles.Palmitoyl-CoA + CarnitineDrosophilaSkeletal muscleF
Smith 2015 Fatty Acid Oxidation O2k-Network Discussion ForumI have extensively tested FAO in PMFBs using the Palmitoylcarnitine, Palmitoyl-CoA, and L-Carnitine. Protocol: For maximal respiration we make sequential additions of 18 µM PC, 20 µM PCoA, and 5 mM Carnitine. Addition of ADP induces a peak rate of O2 consumption that is not increased with FCCP addition.Palmitoylcarnitine
Palmitoyl-CoA
and L-Carnitine
OXPHOS
ET
F
Sparks 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe routinely (up to 4 human and mouse studies) measure respiration supported by glycolytic and fatty acid substrates using SUIT protocols in permeabilized muscle fibers. We always use palmitoylcarnitine. In the past, in Dr. Schrauwen’s lab, we used octanoate to titrate for fatty acid induced uncoupling.Palmitoylcarnitine
Octanoate
Human
Mouse
Skeletal muscleF
Trifunovic 2015 Fatty Acid Oxidation O2k-Network Discussion ForumPalmitoylcarnitine works better than Octanoylcarnitine.Octanoylcarnitine
Palmitoylcarnitine
HeartF
Van Schaardenburgh 2015 Fatty Acid Oxidation O2k-Network Discussion ForumIn my studies on patients with intermittent claudication we used Octanoylcarnitine in the SUIT protocol described by Pesta and you.OctanoylcarnitineF
Veksler 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe studied fatty acid oxidation in permeabilised cardiac and skeletal muscle fibers for 15 years. We use palmitoylcarnitine, palmitoyl-CoA or octanoatePalmitoylcarnitine
Palmitoyl-CoA
Octanoate
Heart
Skeletal muscle
F
Villena 2015 Fatty Acid Oxidation O2k-Network Discussion ForumC14-labelled palmitic acidFatF
Votion 2015 Fatty Acid Oxidation O2k-Network Discussion ForumVery limited (only preliminary tests on mice) but satisfying. I have planned new experiments on equine muscle tissue in 2016.Octanoylcarnitine
Palmitoylcarnitine
Octanoate
Acetylcarnitine
Palmitoyl CoA (+ Carnitine)
MouseF
Weber 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitineHuman
Mouse
GenitalF
West 2015 Fatty Acid Oxidation O2k-Network Discussion ForumOctanoylcarnitineF
Wohlwend 2015 Fatty Acid Oxidation O2k-Network Discussion ForumFatty acid oxidation in permeabilized muscle fibers has worked well for us in both human and rat studies. Typically we use Octanoylcarnitine and Malate for skeletal muscle and a combination of Octanoylcarnitine plus Palmitylcarnitine and Malate for human heart tissue. These protocols have given us a good insight into Beta oxidation.Octanoylcarnitine + Malate
Octanoylcarnitine + Palmitoylcarnitine + Malate (for heart)
Human
Rat
Skeletal muscleF
Wright 2015 Fatty Acid Oxidation O2k-Network Discussion ForumWe do include octanoylcarnitine in the initial titration of substrates for our SUIT protocols typically, but do not measure its individual contribution to OXPHOS.OctanoylcarnitineF
Wuest 2015 Fatty Acid Oxidation O2k-Network Discussion ForumCompared with just glucose oxidation, the max OXPHOS/ET capacity was slightly (but significantly) lower with PC & Oct. Protocol: PC, M, ADP, OCT, G, P, SUC, (FCCP, rat), ROT, AA. The concentrations were the same as in the blue book.Palmitoylcarnitine + OctanoylcarnitineRatHeartOXPHOS
ET
F
N
S
NS
ROX

FA and cell toxicity

I am working at the University of Colorado with Drs Paul MacLean and Matthew Jackman who acquired two OROBOROS this year. Following our collaborators advice (Dr Donal O'Gorman at Dublin City Uni), we are following your protocol to measure fat oxidation. Contrary to results in the literature and from Dr O'Gorman's lab, as soon as we add L-palmitoylcarnitine, the cells die. 2 uM only of L-PC are sufficient to reach a respiration of zero. Adding any other substrates does not allow an increase in O2 consumption. Our local collaborator Dr Jane Reush is encountering the same issue and therefore only use octanoyl carnitine. Octanoyl carnitine does not alter mitochondria/cell. Following advices from Dr Neufer (North Carolina), we added some carnitine. This did not allow maintaining the integrity of the cell. (1)

Because we are in altitude (1600m), we need to use a piO2 of 200-220 (instead of 160) in basal and of 350 (instead of 300) in maximal. We are wondering if this higher piO2 could promote the generation of ROS species or oxidative stress that would kill the mitochondria/cell when adding L-PC? (2)

Audrey (2015-05-14)
O2k-Network: US CO Aurora Jackman MR
Audrey Bergouignan, PhD
Anschutz Health and Wellness Center Division of Endocrinology, Metabolism and Diabetes
University of Colorado
E [email protected]
http://www.anschutzwellness.com/​


Response

  1.  »Fatty acid oxidation#FAO and HRR
  2. When using permeabilized muscle fibers, we have to increase oxygen levels to nonphysiological hyperoxia to avoid oxygen diffusion limitation to the fibers. Although high oxygen levels stimulate ROS production, mitochondria remain functional during times of measurement up to 3 hours. (Gnaiger E, 2015-05-15)
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