Flux through Fatty-acid Oxidation Alters Redox State in Muscle
Cody D. Smith1,2, Chien-Te Lin1, Lauren R. Reese1,2, Cheryl A. Smith1, Irwin J. Kurland3, and P. Darrell Neufer1,2
1East
Carolina Diabetes and Obesity Institute and 2Department of Physiology, East Carolina University, Greenville, NC, USA
3Albert Einstein College of Medicine, Bronx, NY, USA
Results:
Abstract:
Accumulation of myocellular lipid intermediates due to lipid
overload and incomplete mitochondrial fatty acid oxidation (FAO)
have been associated with insulin resistance in muscle. Based
on these findings it has been suggested that therapeutic
acceleration of FAO might alleviate insulin resistance6. According
to principles of mitochondrial bioenergetics, accelerating FAO, in
the absence of energetic demand, will increase the reducing
pressure within the electron transport system (ETS) and
subsequently increase the rate of mitochondrial H2O2 production,
a factor known to cause insulin resistance1, 3. Muscle-specific
transgenic
peroxisome
proliferator-activated
receptor-α
(MCKPPAR) mice are characterized by elevated rates of FAO
and reduced glucose tolerance and insulin sensitivity2.
Permeabilized muscle fiber bundles (PmFbs) prepared from the
white gastrocnemii of MCKPPAR mice had elevated rates of state
4 respiration (JO2) supported solely by fatty acid substrates
compared to wild-type (WT) controls (28.5±2.1 vs. 12.9±0.8 pmol
O2/sec/mg dry wt; mean±SEM; p<0.001) as well as elevated
mitochondrial
membrane
potential
(ΔΨm)
measured
simultaneously (-149.7±1.7 vs. -124.0±5.0 mV; p<0.001),
suggesting increased reducing pressure within the ETS.
Consistent with elevated ΔΨm and in parallel experiments,
PmFbs from MCKPPAR muscle had increased rates of H2O2
production compared to WT (10.4±0.7 vs. 5.8±1.0 pmol/min/mg
dry wt; p<0.01). In PmFbs prepared from homozygous fatty-liver
dystrophy (fld) mouse muscle, another model of accelerated FAO
in skeletal muscle4 and insulin resistance5, state 4 lipid-supported
JO2 and H2O2 production were elevated compared to controls as
well (p<0.05 and p<0.001, respectively). Taken together, these
data suggest elevated FAO flux increases mitochondrial reducing
pressure and H2O2 production, consistent with H2O2 induced
oxidative shifts in cellular redox state linked to high-fat diet
induced insulin resistance. NIH DK096907
Figure 1: Increased flux through FAO is associated with elevated ΔΨm and JH2O2 in MCKPPAR mouse
skeletal muscle.
A.
B.
A) JO2 and ΔΨm were measured simultaneously in PmFbs prepared from white gastroc (WG) and supported by 20µM Palmitoyl-CoA (PCoA), 18µM
Palmitoyl-Carnitine (PC), and 5mM Carnitine (Carn) under state 4 conditions. B) In parallel experiments, JH2O2 was measured using the Amplex
UltraRed/HRP system of H2O2 detection. C&D) JO2 and JH2O2 were measured in PmFbs prepared from red gastroc (RG). Substrates identified on the
x-axis were added in succession. Data are mean ± SEM; * p < 0.05 compared to WT; n=7mice/group.
Figure 2. Increased flux through FAO is associated with elevated JH2O2 in fld-mouse skeletal muscle.
JO2 and JH2O2 were measured in PmFbs prepared from RG (A) and WG (B). Substrates identified on the x-axis were added in succession. Data are
mean ± SEM; * p < 0.05 compared to HET; n=7-8 mice/group.
Objective and Methods:
The objective of this work was to determine whether
accelerated flux through FAO is associated with an elevated
ΔΨm and rate of H2O2 producing potential (JH2O2) in skeletal
muscle.
Figure 3. Increased total GSH and GSSG in MCKPPAR muscle, and increased reduced and oxidized
Prx3 in fld-HOM muscle.
Permeabilized muscle fiber bundle (PmFb) preparation from portions of
dissected red and white gastroc (R/WG) tissue:
5 mm
Model Systems:
• MCKPPAR mice vs. WT littermates, C57Bl/6NJ strain
• Homozygous (HOM) fld mice vs. heterozygous (HET) littermates, BALB/c
strain
• All mice were male, fed a 10% (kcal) fat diet, and sacrificed at 18-20 wks.
Experimental Endpoints:
• High resolution respirometry (JO2)
• Mitochondrial membrane potential
(ΔΨm)
• H2O2 producing potential (JH2O2)
• Reduced/Oxidized Glutathione
(GSH/GSSG)
• Reduced, oxidized dimer, and
oxidized decamer Peroxiredoxin-3
(Prx3)
A) Frozen tibialis anterior (TA) and extensor digitorum longus (EDL) muscle from MCKPPAR and WT mice were used to measure glutathione levels. Homogenization
buffer was bubbled with N2(g) to deplete soluble O2. GSSG samples contained the alkylating agent, M2VP to quench all free thiols. The resulting GSH/GSSG ratio is also
presented. B) Levels of reduced, oxidized dimer, and oxidized decamer Prx3 (Prx3Red, Prx3Oxi, Prx3Deca, respectively) were measured in fld-HOM/HET EDL homogenates
by non-reducing Western blot analysis. A representative blot is presented. β-ME: β-mercaptoethanol treated positive control. Data are mean ± SEM; * p < 0.05
compared to WT or HET; n=7-9 mice/group.
Conclusions:
1. Accelerated flux through FAO is associated with
increased mitochondrial reducing pressure (ΔΨm)
and rate of oxidant production (JH2O2).
2. Increased FAO flux that is not driven by energy
demand creates conditions that promote, rather
than reverse insulin resistance.
References:
1. Ethan J. Anderson, Mary E. Lustig, Kristen E. Boyle, Tracey L. Woodlief, Daniel A. Kane, Chien-Te Lin, Jesse W.
Price, III, Li Kang, Peter S. Rabinovitch, Hazel H. Szeto, Joseph A. Houmard, Ronald N. Cortright, David H.
Wasserman, and P. Darrell Neufer, 'Mitochondrial H2o2 Emission and Cellular Redox State Link Excess Fat Intake
to Insulin Resistance in Both Rodents and Humans', Journal of Clinical Investigation, 119 (2009), 573-81.
2. B. N. Finck, C. Bernal-Mizrachi, D. H. Han, T. Coleman, N. Sambandam, L. L. LaRiviere, J. O. Holloszy, C. F.
Semenkovich, and D. P. Kelly, 'A Potential Link between Muscle Peroxisome Proliferator- Activated ReceptorAlpha Signaling and Obesity-Related Diabetes', Cell Metab, 1 (2005), 133-44.
3. N. Houstis, E. D. Rosen, and E. S. Lander, 'Reactive Oxygen Species Have a Causal Role in Multiple Forms of
Insulin Resistance', Nature, 440 (2006), 944-8.
4. J. Phan, and K. Reue, 'Lipin, a Lipodystrophy and Obesity Gene', Cell Metabolism, 1 (2005), 73-83.
5. K. Reue, P. Xu, X. P. Wang, and B. G. Slavin, 'Adipose Tissue Deficiency, Glucose Intolerance, and Increased
Atherosclerosis Result from Mutation in the Mouse Fatty Liver Dystrophy (Fld) Gene', Journal of Lipid Research,
41 (2000), 1067-76.
6. G. I. Shulman, 'Cellular Mechanisms of Insulin Resistance', Journal of Clinical Investigation, 106 (2000), 171-76.