Functional and animal studies of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), a fatty acid beta-oxidation enzyme involved in congenital hyperinsulinism of infancy

Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) is a mitochondrial enzyme involved in fatty acid β-oxidation and the regulation of β-cell insulin secretion. Its role in insulin secretion became apparent in 2001 when the first report linking SCHAD deficiency with congenital hyperinsulinism of infancy (CHI) was published. Since then research into the mechanism of SCHAD-CHI has led to the view that SCHAD serves a β-cell specific function and that the mechanism involves inhibition of glutamate dehydrogenase. In this thesis, we studied functional differences of pathogenic and non-pathogenic SCHAD missense variants, investigated the β-cell specificity of SCHAD deficiency in an animal model, and sought to identify novel interaction partners of the protein. In Paper 1, we found that missense SCHAD variants occurring in datasets from human populations mostly behaved like the wild type protein, whereas CHI-associated SCHAD variants had altered properties with regard to protein stability, enzymatic function and interaction with GDH. Four of the seven studied CHI variants showed reduced protein expression when expressed in HEK293 cells. The remaining three pathogenic variants had stable expression but reduced enzymatic activity and binding to GDH. In Paper II, we generated conditional SCHAD knockout mice and studied the cell-type specific effects of SCHAD deficiency in β-cells and hepatocytes. We noted that the SCHAD protein was highly expressed in pancreatic β- and -cells, but virtually absent from the α-cells. The mice harboring a β-cell-specific deficiency in SCHAD expression were hypoglycemic under various conditions, and their islets were sensitive to amino acid-stimulated insulin secretion. Analysis of the transcriptome of isolated SCHAD knockout islets identified global changes in expression of genes involved in metabolism and β-cell identity. Overall, this study strengthened the hypothesis that SCHAD serves a specific function in the β-cells and that β-cell SCHAD deficiency is sufficient to cause hypoglycemia. In Paper III, we sought to identify novel protein interactions partners of SCHAD by performing a yeast two-hybrid screen in a library from human islets of Langerhans. We identified keratin 8 (K8) as a putative binding partner and found additional evidence of an interaction by co-immunoprecipitation experiments. We also analyzed SCHAD expression in a K8 knockout mouse and K8 expression in the SCHAD knockout mouse. However, the lack of one potential interaction partner did not affect expression of the other, except when K8 knockout mice were fed a ketogenic diet. The dietary challenge resulted in an upregulation of SCHAD in the pancreas of WT animals and this was blunted in the absence of K8. More experiments need to be carried out in order to explore the possible biological relevance of the proposed SCHAD-K8 interaction. In summary, this thesis increased our knowledge on the impact of missense variants on SCHAD function and may therefore aid in the clinical evaluation of rare variants found in patients with phenotypes related to β-cell function. We developed a new mouse model for SCHAD-CHI, which highlighted the importance for SCHAD expression in the β-cell as well as the heterogeneous expression of the enzyme in islet cell populations. Lastly, we found evidence for a novel and surprising protein interaction of SCHAD with K8 that remains to be further validated.Doktorgradsavhandlin