The gut microbiota as a contributing factor to antipsychotic-induced weight gain and metabolic dysfunction

Schizophrenia represents one of the world’s most devastating illnesses due to its often lifelong course and debilitating nature. The treatment of schizophrenia has vastly improved over recent decades with the discovery of several antipsychotic compounds; however these drugs are not without adverse effects that must be addressed to maximize their therapeutic value. Newer, atypical, antipsychotics are associated with a compilation of serious metabolic side effects including weight gain, insulin resistance, fat deposition, glucose dysregulation and ensuing co-morbidities such as type II diabetes mellitus. The mechanisms underlying these side effects remain to be fully elucidated and adequate interventions are lacking. Further understanding of the factors that contribute these side effects is therefore required in order to develop effective adjunctive therapies and to potentially design antipsychotic drugs in the future with reduced impact on the metabolic health of patients. We investigated if the gut microbiota represented a novel mechanism contributing to the metabolic dysfunction associated with atypical antipsychotics. The gut microbiota comprises the bacteria that exist symbiotically within the gastrointestinal tract, and has been shown in recent years to be involved in several aspects of energy balance and metabolism. We have demonstrated that administration of certain antipsychotics in the rat results in an altered microbiota profile and, moreover, that the microbiota is required for the full scale of metabolic dysfunction to occur. We have further shown that specific antibiotics can attenuate certain aspects of olanzapine and risperidone–induced metabolic dysfunction, in particular fat deposition and adipose tissue inflammation. Mechanisms underlying this novel link appear to involve energy utilization via expression of lipogenic genes as well as reduced inflammatory tone. Taken together, these data indicate that the gut microbiota is an important factor involved in the myriad of metabolic complications associated with antipsychotic therapy. Furthermore, these data support the future investigation of microbial-based therapeutics for not only antipsychotic-induced weight gain but also for tackling the global obesity epidemic.

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.

Multi-parametric metabolic assessment of cells under stress conditions

Glycolysis, glutaminolysis, the Krebs cycle and oxidative phosphorylation are the main metabolic pathways. Exposing cells to key metabolic substrates (glucose, glutamine and pyruvate); investigation of the contribution of substrates in stress conditions such as uncoupling and hypoxia was conducted. Glycolysis, O2 consumption, O2 and ATP levels and hypoxia inducible factor (HIF) signalling in PC12 cells were investigated. Upon uncoupling with FCCP mitochondria were depolarised similarly in all cases, but a strong increase in respiration was only seen in the cells fed on glutamine with either glucose or pyruvate. Inhibition of glutaminolysis reversed the glutamine dependant effect. Differential regulation of the respiratory response to FCCP by metabolic environment suggests mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function. At reduced O2 availability (4 % and 0 % O2), cell bioenergetics and local oxygenation varied depending on the substrate composition. Results indicate that both supply and utilisation of key metabolic substrates can affect the pattern of HIF-1/2α accumulation by differentially regulating iO2¬, ATP levels and Akt/Erk/AMPK pathways. Inhibition of key metabolic pathways can modulate HIF regulatory pathways, metabolic responses and survival of cancer cells in hypoxia. Hypoxia leads to transcriptional activation, by HIF, of pyruvate dehydrogenase (PDH) kinase which phosphorylates and inhibits PDH, a mitochondrial enzyme that converts pyruvate into acetyl-CoA. The levels of PDH (total and phosphorylated), PDH kinase and HIF-1α were analysed in HCT116 and HCT116 SCO2-/- (deficient in complex IV of the respiratory chain) grown under 20.9 % and 3 % O2. Data indicate that regulation of PDH can occur in a manner independent of the HIF-1/PDH kinase 1 axis, mitochondrial respiration and the demand for acetyl-CoA. Collectively these results can be applied to many diseases; reduced nutrient supply and O2 during ischemia/stroke, hypoglycaemia in diabetes mellitus and cancer associated changes in uncoupling protein expression levels.