Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARα) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARα ligands. Here, we characterized a binding site for PPARα in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARα binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1α) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1α did not enhance CPT-1A transcription through the PPARα binding site in the second intron. Following knockdown of PGC-1α with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARα and PGC-1α stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK) inhibits its activity. The expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene is increased in fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. Transcription of the PDK4 gene is elevated by glucocorticoids and inhibited by insulin. In this study, we have investigated the factors involved in the regulation of the PDK4 gene by these hormones. Glucocorticoids stimulate PDK4 through two glucocorticoid receptor (GR) binding sites located more than 6,000 base pairs upstream of the transcriptional start site. Insulin inhibits the glucocorticoid induction in part by causing dissociation of the GR from the promoter. Previously, we found that the estrogen related receptor alpha (ERRα) stimulates the expression of PDK4. Here, we determined that one of the ERRα binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRα binding sites. FoxO1 participates in the glucocorticoid induction of PDK4 and the regulation of this gene by insulin. Our data demonstrate that glucocorticoids and insulin each modulate PDK4 gene expression through complex hormone response units that contain multiple factors.

Striking individual differences in severity of group A streptococcal (GAS) sepsis have been noted, even among patients infected with the same bacterial strain. We had provided evidence that HLA class II allelic variation contributes significantly to differences in systemic disease severity by modulating host responses to streptococcal superantigens. Inasmuch as the bacteria produce additional virulence factors that participate in the pathogenesis of this complex disease, we sought to identify additional gene networks modulating GAS sepsis. Accordingly, we applied a systems genetics approach using a panel of advanced recombinant inbred mice. By analyzing disease phenotypes in the context of mice genotypes we identified a highly significant quantitative trait locus (QTL) on Chromosome 2 between 22 and 34 Mb that strongly predicts disease severity, accounting for 25%–30% of variance. This QTL harbors several polymorphic genes known to regulate immune responses to bacterial infections. We evaluated candidate genes within this QTL using multiple parameters that included linkage, gene ontology, variation in gene expression, cocitation networks, and biological relevance, and identified interleukin1 alpha and prostaglandin E synthases pathways as key networks involved in modulating GAS sepsis severity. The association of GAS sepsis with multiple pathways underscores the complexity of traits modulating GAS sepsis and provides a powerful approach for analyzing interactive traits affecting outcomes of other infectious diseases.

Variation in responses to pathogens is influenced by exposure history, environment and the host's genetic status. We recently demonstrated that human leukocyte antigen class II allelic differences are a major determinant of the severity of invasive group A streptococcal (GAS) sepsis in humans. While in-depth controlled molecular studies on populations of genetically wellcharacterized humans are not feasible, it is now possible to exploit genetically diverse panels of recombinant inbred BXD mice to define genetic and environmental risk factors. Our goal in this study was to standardize the model and identify genetic and nongenetic covariates influencing invasive infection outcomes. Despite having common ancestors, the various BXD strains (n strains ¼ 33, n individuals ¼ 445) showed marked differences in survival. Mice from all strains developed bacteremia but exhibited considerable differences in disease severity, bacterial dissemination and mortality rates. Bacteremia and survival showed the expected negative correlation. Among nongenetic factors, age -but not sex or weight -was a significant predictor of survival (P ¼ 0.0005). To minimize nongenetic variability, we limited further analyses to mice aged 40-120 days and calculated a corrected relative survival index that reflects the number of days an animal survived post-infection normalized to all significant covariates. Genetic background (strain) was the most significant factor determining susceptibility (Pp0.0001), thus underscoring the strong effect of host genetic variation in determining susceptibility to severe GAS sepsis. This model offers powerful unbiased forward genetics to map specific quantitative trait loci and networks of pathways modulating the severity of GAS sepsis.