triheptanoin as a novel therapy for metabolic myopathies ... · triheptanoin as a novel therapy for...
TRANSCRIPT
L. BorgiaL. Borgia11, S.J. Valberg, M.E. McCue, J. , S.J. Valberg, M.E. McCue, J. Pagan, and C. Roe Pagan, and C. Roe
1 1 University of Minnesota College of Veterinary Medicine St Paul MUniversity of Minnesota College of Veterinary Medicine St Paul MNN
Triheptanoin as a novel therapy for Triheptanoin as a novel therapy for metabolic metabolic myopathiesmyopathies and muscular and muscular
dystrophiesdystrophies
IntroductionIntroduction
Amylase-resistant abnormal polysaccharide in cells
Polysaccharide Storage Myopathy (PSSM), a genetic neuromuscular disease found in horses
Increased synthesis of glycogen and accumulation of abnormal polysaccharide in muscleIncreased synthesis of glycogen (GYS1).
Cell damage and impaired Cell damage and impaired energy metabolism may deplete energy metabolism may deplete CAC intermediatesCAC intermediatesTriheptanoin (C7) shown to Triheptanoin (C7) shown to provide C3 to replenish depleted provide C3 to replenish depleted CAC intermediatesCAC intermediatesAbsorption of C7 occurs in PSSM Absorption of C7 occurs in PSSM horseshorses
C7 C5 propionyl CoA m-malonyl CoA+ acetyl CoA
beta oxidation of C18
Horses consuming TH or CO
TH CO 0.00
0.01
0.02
0.03
C7μ
M
malate
succinatesuccinyl-CoA
α-ketoglutarate
isocitrate
oxaloacetate
fumarate
citrate
acetyl-CoA
Triheptanoin (TH) used to treat MCAD deficiency in humansWider use suggested for similar metabolic myopathies in humans
Animal Model: PSSMAnimal Model: PSSM
Current PSSM TreatmentCurrent PSSM Treatment
Post exercise serum creatine kinase activity (CK) affected by diet:Therapeutic diet– Low starch– High long chain fat corn oil (CO)
linoleic (C18:2) and oleic acid (C18:1)
Hypothesis/ObjectivesHypothesis/ObjectivesHypothesis: Provision of triheptanoin (C7), which may replete CAC intermediates, will be more effective than C18 in ameliorating signs of rhabdomyolysis and reducing serum CK activity.rhabdomyolysis and reducing serum CK activity.
Rationale: rhabdomyolysis in may result in loss of CAC intermediates impairing cellular energy metabolism
Objectives: 1. Objectives: 1. To determine if serum CK activity following submaximal To determine if serum CK activity following submaximal exercise is lower on triheptanoin vs. corn oil. exercise is lower on triheptanoin vs. corn oil.
2.2. To determine if substrate availability during submaximal exerciTo determine if substrate availability during submaximal exercise is se is impacted by provision of triheptanoin or corn oil.impacted by provision of triheptanoin or corn oil.
DesignDesign
Forage base:Forage base:–– 1.5% of 1.5% of bwbw in grass hayin grass hay–– 0.5 kg ration balancer0.5 kg ration balancer
Oil diets:Oil diets:–– 1.5 kg hay cubes 1.5 kg hay cubes –– 1.5 ml/kg/day TH or CO1.5 ml/kg/day TH or CO–– Divided 3 feeding/dayDivided 3 feeding/day
IsocaloricIsocaloric diets providing 25 diets providing 25 MCalMCal/day /day corn oil or triheptanoincorn oil or triheptanoin
Randomized crossRandomized cross--over over design with 2 blocks design with 2 blocks Each block lasted 3 wksEach block lasted 3 wks1 wk washout between 1 wk washout between blocksblocksHorses exercised 5 Horses exercised 5 days/wk on each blockdays/wk on each blockDaily blood samples 4 hr Daily blood samples 4 hr after exercise for serum after exercise for serum CK activityCK activity
Materials and Methods Materials and Methods -- HorsesHorses
8 horses previously 8 horses previously diagnosed with PSSM diagnosed with PSSM based on amylase based on amylase resistant polysaccharideresistant polysaccharide6 mares, 1 gelding , 1 colt6 mares, 1 gelding , 1 colt2 2 –– 14 yrs of age (6.25 14 yrs of age (6.25 ±±3.9 yrs3.9 yrs))Stall rested for a minimum Stall rested for a minimum of 3 wks prior to start of of 3 wks prior to start of treadmill exercisetreadmill exercise
Materials and MethodsMaterials and Methods--Starch dietStarch dietIndividual treadmill exercise protocol established Individual treadmill exercise protocol established
25 25 MCalMCal/day /day –– Forage Base Forage Base
1.5% 1.5% bwbw grass haygrass hay0.5 kg ration 0.5 kg ration balancerbalancer
–– Concentrate per Concentrate per 500kg500kg
1.7 kg sweet feed1.7 kg sweet feed1 kg rice bran1 kg rice bran
Exercise toleranceExercise tolerance–– Week 1: Week 1:
intervals walk and intervals walk and trot (2 min) trot (2 min)
–– Week 2 & 3:Week 2 & 3:Establish individual Establish individual exercise protocol exercise protocol (daily treadmill (daily treadmill exercise target)exercise target)
Materials and Methods Materials and Methods -- TreadmillTreadmill
Same individual exercise protocol Same individual exercise protocol across blocksacross blocksSame handler for each horseSame handler for each horseHandlers blinded to dietHandlers blinded to dietExercise terminated prematurely Exercise terminated prematurely if horse became stiff, excessive if horse became stiff, excessive sweating or unable to keep pace sweating or unable to keep pace Subsequent exercise session was Subsequent exercise session was reduced to 2 min if overt signs of reduced to 2 min if overt signs of ER or CK> 50,000 U/L ER or CK> 50,000 U/L Exercise duration gradually Exercise duration gradually increased increased
Materials and Methods Materials and Methods -- Exercise testExercise test
Exercise testExercise testLast day of each Last day of each block. 2 min block. 2 min intervals walk intervals walk (1.9 (1.9 m/sm/s) trot ) trot (3(3--4.7 4.7 m/sm/s) for ) for 15 min15 minBlood collected Blood collected pre/post pre/post exercise via exercise via jugular catheter jugular catheter
Muscle needle Muscle needle biopsies biopsies obtained obtained pre/immediately pre/immediately post exercise post exercise through same through same incision, snap incision, snap frozen in liquid Nfrozen in liquid NSerum CK Serum CK measured 4 hr measured 4 hr post exercisepost exercise
Materials and Methods Materials and Methods -- Exercise testExercise test
Statistical AnalysisStatistical AnalysisCK CK –– repeated measures ANOVA log repeated measures ANOVA log
CKCK–– Not all horses achieved daily Not all horses achieved daily
exercise target. exercise target. ANCoVAANCoVA log CK log CK using % daily exercise achieved using % daily exercise achieved as covariant.as covariant.
Pre and post exercise blood Pre and post exercise blood samplessamples–– Glucose, insulin, NEFA, Glucose, insulin, NEFA,
acylcarnitines acylcarnitines –– 2 way ANOVA2 way ANOVA
Pre exercise glycogen Pre exercise glycogen (muscle)(muscle)–– t testt test
Reported as mean Reported as mean ±± standard standard deviationdeviation
Results: CKResults: CKMean CK activityMean CK activityTH > grainTH > grainTH > COTH > COCO < grainCO < grain
Exercise ToleranceExercise Tolerance–– THTH
37 days horses < target 37 days horses < target (84% achieved)(84% achieved)
–– COCO5 days horses < target 5 days horses < target (99% achieved)(99% achieved)
Mean CK values
Grain Triheptanoin Corn oil0
1000
2000
3000
4000
5000
6000
CK
(U/L
)
a
b
c
Mean daily CK values
0 4 8 12 16100
1000
10000
graintriheptanoin
corn oil
Day
CK
(U/L
)
Results: SubstratesResults: Substrates
Glycogen
TH CO0
100200300400500600700800
Diet
mm
ol/k
g
GlycogenGlycogen–– Diet effectDiet effect
No difference No difference between blocksbetween blocks
Results: Plasma Results: Plasma CarnitinesCarnitines
C18:2 acylcarnitineC18:2 acylcarnitine–– Diet effectDiet effect
TH<COTH<CO
–– Exercise Effect Exercise Effect Pre < PostPre < Post
C3 acylcarnitineC3 acylcarnitine–– Diet effectDiet effect
TH>COTH>CO
–– Exercise Effect Exercise Effect NoneNone
C18:2 acylcarnitine
TH pre TH Post CO Pre CO Post0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
C3 acylcarnitine
TH CO0
1
2
Diet
μM
pre
prepost
post
Results: SubstratesResults: Substrates
Diet EffectDiet Effect–– Insulin Insulin
TH > CO TH > CO CO < grainCO < grain
–– GlucoseGlucoseNo difference No difference
Exercise Effect Exercise Effect –– Glucose and InsulinGlucose and Insulin
Pre > Post exercisePre > Post exercise
Insulin pre and post exercise
Grain TH CO0
5
10
15
20
25
30
35
prepre
prepost post post
Diet
μm
ol/m
l
Results: SubstratesResults: Substrates
NEFANEFA–– Diet effectDiet effect
TH<COTH<COGrain<C0Grain<C0
–– Exercise Effect Exercise Effect No differenceNo difference
NEFA Pre/Post Exercise
Grain TH CO0.00
0.05
0.10
0.15
postpost
post
pre pre
pre
Dietm
mol
/l
DiscussionDiscussion
Plasma acylcarnitine concentrations suggested Plasma acylcarnitine concentrations suggested that TH was absorbed and metabolized to C3, C5 that TH was absorbed and metabolized to C3, C5 potentially providing CAC intermediates.potentially providing CAC intermediates.Rather than a beneficial effect, TH exacerbated Rather than a beneficial effect, TH exacerbated muscle damage during exercise as indicated by muscle damage during exercise as indicated by serum CK activity.serum CK activity.Further, TH caused exercise intolerance as Further, TH caused exercise intolerance as indicated by inability to achieve daily exercise indicated by inability to achieve daily exercise target. target. Detrimental effect of TH was not related to higher Detrimental effect of TH was not related to higher muscle glycogen concentrationsmuscle glycogen concentrations
DiscussionDiscussionTH produced plasma insulin TH produced plasma insulin concconcsimilar to grain (> CO). similar to grain (> CO). –– Increased CAC intermediates Increased CAC intermediates
““secretagoguessecretagogues”” may induce insulin may induce insulin secretion by pancreassecretion by pancreas
Activation of lipoprotein lipase by Activation of lipoprotein lipase by insulin may explain low NEFA insulin may explain low NEFA concentrations on grain and TH.concentrations on grain and TH.Lack of beneficial effect of TH Lack of beneficial effect of TH may be related to low NEFA and may be related to low NEFA and inadequate long chain fats inadequate long chain fats available for metabolism.available for metabolism.
DiscussionDiscussionCorn oil had a beneficial effect Corn oil had a beneficial effect and lowered serum CK activity.and lowered serum CK activity.Lower plasma insulin on CO Lower plasma insulin on CO diet resulted in higher diet resulted in higher concentrations of plasma concentrations of plasma NEFAsNEFAs available for available for ββ--oxidation oxidation during exercise.during exercise.Plasma C18:2 and C18:1 Plasma C18:2 and C18:1 acylcarnitine increased with acylcarnitine increased with exercise, suggesting dietary exercise, suggesting dietary long chain fats were long chain fats were metabolized by PSSM horses.metabolized by PSSM horses.
ConclusionConclusion
TH may be of benefit as a therapeutic diet TH may be of benefit as a therapeutic diet for carefully selected metabolic for carefully selected metabolic myopathiesmyopathies–– MCADsMCADs–– PompePompe diseasedisease–– PatientPatient’’s endocrine status should be evaluateds endocrine status should be evaluated
TH may not be of benefit to those with TH may not be of benefit to those with diabetes or those needing LC fats for diabetes or those needing LC fats for energy metabolismenergy metabolism
Next StepsNext Steps
Analysis of the effect of starch, sugar, and Analysis of the effect of starch, sugar, and fructanfructan concentrations in forages (hay) on concentrations in forages (hay) on horses with PSSMhorses with PSSMContinue to explore mechanisms of this Continue to explore mechanisms of this disorderdisorder
AcknowledgementsAcknowledgements
Michelle Michelle LucioLucio, April Holden, Krista Steffenhagen, April Holden, Krista SteffenhagenSupported by Office of Clinical and Translational Supported by Office of Clinical and Translational Research, Morris Animal Foundation, Eric Research, Morris Animal Foundation, Eric MarzolfMarzolfMuscular Dystrophy grant, and University of Muscular Dystrophy grant, and University of Minnesota Equine Center. Grand opening October Minnesota Equine Center. Grand opening October 15 at 2pm!15 at 2pm!