Dietary fat avoidance in Acads mutant mice

1. Dietary fat avoidance in Acads mutant mice B.K. (Smith) Richards Pennington Biomedical Research Center

2. 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.

3. 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 JacksonLaboratory. • 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.

4. Tafti et al. Nat Genet, 2003

5. 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)

6. 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).

7. Fat preference across mouse strains(Smith, Andrews, & West, Am J Physiol 278, 2000)

8. Defect in fatty acid oxidation lowers self-selected fat intake, but not total calories (Smith Richards et al, Am J Physiol 286: R311-R319, 2004)

9. 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.

10. 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).

11. 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).

12. Mechanisms? Could fat avoidance be the result of an altered orosensory response?

13. 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)

14. 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.

15. We hypothesize that gene expression patterns will reveal functionally important brain regions and hepatic pathways mediating the behavioral feeding response to dietary fat.

16. Specific Aims: • 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.

17. 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 nucleus accumbens • amygdala brainstem (NTS) • hypothalamus ventral tegmental area • Test for enrichment of nuclei-specific markers to establish regions. • Validate the observed expression differences using real-time qPCR.

18. 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