Carruthers, Jack Edward; (2023) The role of systemic and hepatic glycerol metabolism in metabolic disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Obesity is multifactorial chronic disease that is associated with a wide-range of health complications including insulin resistance (IR) that can lead to type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD) that encompasses non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and cirrhosis. Dysregulation of tissue-level and systemic metabolism characterises these conditions. In IR, for example, there is a pathological failure to suppress gluconeogenesis (GNG) in response to insulin, which can result in raised endogenous glucose production (EGP). In NAFLD, there is excessive deposition of triacylglycerols (TAGs) in the liver. Glycerol, a three-carbon metabolite, is placed at the cross-roads of both lipid and carbohydrate metabolism. It is converted by glycerol kinase (GyK) to glycerol-3-phosphate (G3P) which can provide carbons for glucose production and for the esterification of fatty acids to TAGs. As a result, it may be implicated in the pathogenesis of both obesity-related IR and NAFLD. Evidence from human studies suggests that glycerol turnover correlates with glycerol-GNG in patients with obesity and T2DM, whilst the contribution of glycerol to circulating TAGs is augmented in patients with NAFLD. Further, ageing may modulate the use of glycerol as a carbon source for metabolic processes. Interestingly, ageing per se is associated with IR and is considered a risk factor for the development of obesity and NAFLD. Despite this correlative evidence, the role of glycerol in supporting the pathological changes associated with obesity as it develops over time has never been described. In this study, glycerol metabolism is characterised in a Western diet (WD)-induced obesity mouse model with NAFLD and IR. This model encompasses chronicity of dietary exposure, and therefore ageing, from 12 to 50 weeks of feeding. Obesity and age act in concert to alter glycerol clearance from the circulation in response to 24 hr fasting and 4 hr refeeding. In addition, WD-feeding increases glycerol turnover and the partitioning of glycerol into systemic glucose and hepatic TAG as shown by the infusion of stable isotopes [U-13C]glucose and [1,3-13C]glycerol. These findings reveal that glycerol is used to support both EGP and hepatic steatosis in ageing, IR and NAFLD. In order to perturb the glycerol metabolic pathway, GyK was deleted at the whole-body and hepatic level. Whole-body GyK deletion (GyKKO) increases circulating glycerol, and decreases glycerol turnover, EGP and hepatic TAG esterification. In order to determine the role of hepatic versus extrahepatic GyK in coordinating glycerol metabolism, a liver-specific GyKKO (LGyKKO) was compared to the GyKKO. This revealed that hepatic GyK accounts for the majority of glycerol partitioning into glucose or lipid metabolism. The decrease in EGP was driven by a marked decreased in hepatic glucose production in the LGyKKO, as shown by isolated liver perfusions with GNG substrates and deuterium oxide followed by nuclear magnetic resonance (NMR). Finally, herein is presented evidence that the liver integrates different dietary inputs to overcome GyK deletion and promote NAFLD in WD-fed LGyKKO mice. Infusions of [U-13C]glucose and [U-13C]fructose in WD-fed LGyKKO mice revealed that both dietary glucose and fructose are converted to G3P to compensate for the loss of hepatic GyK. The contribution of fructose to G3P correlates with the amount of fructose in the diet. Several metabolomics methodologies including NMR, 2D-GCMS and lipidomics were used to determine unanticipated consequences of hepatic GyK deletion. This approach has revealed alterations in -oxidation and acylcarnitine biology in LGyKKO mice, whilst simultaneously have highlighted that the WD is the main driver of hepatic metabolic adaptation. This work has implications for the understanding of the role of glycerol in directing the metabolic changes that accompany ageing and obesity pathogenesis.

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