Investigating the potential relevance of SSADH-mediated GABA catabolism outside the CNS for metabolic disease

Anderson, J. (Josephine) (2016) Investigating the potential relevance of SSADH-mediated GABA catabolism outside the CNS for metabolic disease. thesis, Medicine.

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Abstract

Introduction: Metabolism of the inhibitory neurotransmitter Gamma-Aminobutyric acid (GABA) occurs through the synchronous action of GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Several hundred patients are diagnosed with SSADH deficiency primarily suffering from epilepsia among other neurological symptoms. However, these patients appear relatively protected against the development of metabolic syndrome-related disorders. Since GABAergic mechanisms were shown to be active in peripheral tissue as well, we hypothesized that this apparent metabolic protection is conferred by the biological activities of GABA outside the central nervous system. Therefore, the aim of the present study was to investigate the expression of GABA-metabolizing enzymes in peripheral tissues and to provide an initial characterization of potential functional consequences of reduced SSADH expression. Method: A variety of mouse organs, human liver and a number of macrophages and hepatocyte-derived cell lines were analyzed by means of qPCR for the expression of SSADH, and other GABA metabolizing genes. Western blot was preformed to confirm SSADH protein expression. SSADH was downregulated in HepG2 cells using an SSADH specific siRNA and changes in mRNA expression of a number of genes involved in (chol)sterol metabolism were assessed. Results: SSADH was expressed in mouse liver and adrenals, as well as in human liver and the human liver cell lines IHH and HepG2. No expression was found in Kupffer cells, M1 macrophages, M2 macrophages and THP-1 cells. Complete knockdown of SSADH was achieved in HepG2 cells using siRNA transfection. Interestingly, a significantly lower CYP7A1 expression was observed in SSADH silenced cells compared with the scrambled control, while the expression of other genes involved in cholesterol synthesis and uptake remained unaltered. Discussion: This study indicates that SSADH is expressed in hepatocytes where it appears to be functionally associated with the expression of CYP7A1, a key enzyme for bile acid synthesis. Decreased expression of CYP7A1, likely results in a higher relative contribution of CYP8B1 driven bile acid synthesis, which is expected to lead to more cholic acid and subsequently deoxycholic acid (DCA) production. In preclinical models, DCA was shown to reverse high fat diet-induced weight gain and improve glucose tolerance. Thus, more DCA production upon reduced SSADH expression could provide an explanation for the protective effect of SSADH mutations against obesity and insulin resistance. However, this concept needs to be tested in further in vivo studies. Conclusion: This study demonstrates substantial expression of SSADH in hepatocytes and suggests a potential link between GABA and bile acid metabolism, which could explain the decreased incidence of metabolic syndrome-related disease observed in epileptic patients

Item Type:	Thesis (Thesis)
Supervisor name:	Supervisor: and Tietge, Prof. Uwe and Department of Pediatrics, Centre for Liver and University Medical Centre Groningen
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:56
Last Modified:	25 Jun 2020 10:56
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/1694

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