Transcript Document

Dietary fat avoidance
in Acads mutant mice
B.K. (Smith) Richards
Pennington Biomedical Research Center
Enzymes of mitochondrial b-oxidation
Acyl-CoA dehydrogenases (AD) that
catalyze the first step of mitochondrial
fatty acid beta-oxidation:
Very-long-chain (VLCAD)
Long-chain (LCAD)
Medium-chain (MCAD)
Short-chain (SCAD)
These four enzymes differ in their
substrate specificity based on the chain
length of the fatty acids that they process.
Taken from S. Eaton et al., Biochem J, 1996.
Abbreviations: CPT, carnitine palmitoyltransferase;
ETF, electron transfer flavoprotein; ETF: QO;
ETF:ubiquinone oxidoreductase; ETFH: reduced ETF.
Animal Model
•
Functional short-chain acyl-CoA dehydrogenase (SCAD) enzyme is absent in
BALB/cByJ mice due to a spontaneous mutation in Acads (Wood et al., 1989).
• The mutation consists of a 278 bp deletion in the 3’ end of the structural gene
for SCAD.
• The Acads mutation occurred spontaneously between 1981 and 1982 in the
BALB/cByJ production line (Reue & Cohen, 1996), descendents of the BALB/cBy
strain maintained originally by Donald Bailey at the Jackson Laboratory.
• The best SCAD-normal control line for the BALB/cByJ (Acads -/-) strain is the
coisogenic BALB/cBy (Acads +/+).
• A colony of BALB/cByKz.Acads -/- and BALB/cByKz.Acads +/+ mice was
established at the PBRC by Dr. Leslie Kozak.
Tafti et al.
Nat Genet, 2003
Phenotypes
Acads -/- mice appear clinically normal.
Phenotypes:
• accumulation and secretion of fatty acid metabolites in urine
• fasting-induced hypoglycemia
• fatty liver and kidney
• cold intolerance (Guerra et al., 1998)
• slowing of theta oscillations during sleep (Tafti et al., 2003)
• dietary fat avoidance (Smith et al., 2004)
Human SCAD deficiency
 First described in 1987, SCAD deficiency is an autosomal recessive, clinically
heterogeneous disorder with only 22 case reports published so far (van
Maldegem et al., 2006).
 Two common SCAD gene variants (625GA and 511CT) have been
identified and are regarded as susceptibility variations.
 The clinical features range from asymptomatic to short-chain dicarboxylic
aciduria, nonketotic hypoglycemia, and metabolic acidosis.
 The standard treatment of fatty acid oxidation disorders is through nutritional
therapy with a focus on restricting dietary fat intake (typically 30% or less).
NZ B /B 1NJ
A K R/J
0
CA S T / E i
B A LB / cB yJ
129/ J
S WR/J
S JL/ J
CD-1
S P RE T / E i
DB A /2J
C57B L/ 6B yJ
C57B L/ 6J
Perc ent energy from fat
Fat preference across mouse strains
(Smith, Andrews, & West, Am J Physiol 278, 2000)
100
80
60
40
20
Defect in fatty acid oxidation lowers
self-selected fat intake, but not total calories
25
Ac ads -/-
Acads -/-
Ac ads +/+
80
Total daily kcals
Percent energy from fat
100
60
40
20
0
20
15
10
5
0
1
2
3
4
5
Day
6
7
8
9
10
Acads +/+
1
2
3
4
5
6
7
Day
(Smith Richards et al, Am J Physiol 286: R311-R319, 2004)
8
9
10
Acads -/- inbred mouse strain provides a new model
for investigating pathways regulating food intake
and nutrient selection.
This model is particularly relevant because
fatty acid oxidation is thought to be a key factor
in the metabolic control of food intake.
Acads -/- and Acadl -/Impaired oxidation of either SC or LC fatty acids does not inhibit food intake
in a choice paradigm or single HF diet (not shown).
Hypothalamic Nutrient Sensing
Intracerebroventricular (ICV) administration of the LCFA oleic acid
markedly inhibits glucose production and food intake (Obici et al., 2002).
Regulation of food intake by hypothalamic LCFA-CoA levels can occur
through changes in expression or activity of CPT I, changes in AMPK
activity, or inhibition of fatty acid synthase (Lam et al., 2005).
This effect is specific for LCFAs, e.g., ICV administration of the MCFA
octanoic acid does not inhibit food intake or glucose production in the liver
(Obici et al., 2002).
Stimulation of feeding by inhibitors of fatty acid oxidation
Rodents are stimulated to eat when treated systemically with
pharmacological inhibitors that interfere with beta-oxidation of fatty
acids:


Mercaptoacetate (MA) inhibits FAO in the mitochondria
Methyl palmoxirate or emeriamine inhibit carnitine
palmitoyltransferase I (CPT I)
Fat-specific effects:
MA increased carbohydrate or protein intake, but failed to enhance fat
consumption, both in a 3-choice paradigm and when fat was the only
available nutrient source (Singer LK et al,1998).
Mechanisms?
Could fat avoidance be the result of an altered orosensory response?
Acads deficiency does not alter orosensory
response to corn oil in brief-access tests
(Smith Richards et al, Am J Physiol 286: R311-R319, 2004)
Very little information is available concerning the
specific mechanisms by which metabolic or energy status in
the periphery, e.g., liver is transduced into a signal that is
sensed by the nervous system.
The question addressed in this proposal is how
impaired short-chain fatty acid metabolism is translated into
events that affect feeding behavior via the CNS.
We hypothesize that gene expression patterns will reveal
functionally important brain regions and hepatic pathways
mediating the behavioral feeding response to dietary fat.
Specific Aims:
1. To identify the subset of genes activated by the response to fat
intake, in liver and selected brain nuclei of Acads -/- mice using a
genome-wide mouse array.
2. To validate the observed expression differences using real-time
qPCR analysis.
Experimental Design
 Compare gene expression in Acads -/- and Acads +/+ mice after
48 h ingestion of 10% or 58% fat diets (Research Diets, Inc).
 Euthanize twelve mutant and 12 wild-type mice: remove brain and liver,
collect plasma for acylcarnitine analyses.
 Select 3 mice of each strain (biological replicates) and profile
transcriptional differences using the ABI mouse genome microarray (P<0.01).
 Obtain 0.5- or 1-mm coronal brain sections. Tissue samples will be
punched (0.5 mm diameter) bilaterally & RNA extracted from selected nuclei:
pre-frontal cortex
amygdala
hypothalamus
nucleus accumbens
brainstem (NTS)
ventral tegmental area
 Test for enrichment of nuclei-specific markers to establish regions.
 Validate the observed expression differences using real-time qPCR.
Next steps
Continue analyses of liver data & select genes for qPCR validation
Complete metabolic assays (insulin, glucose, acylcarnitines)
Profile gene expression patterns in brain: hypothalamus and NTS