title: Chronic Inflammation: A Link Between Cardiometabolic and Mood Disorders?
Creators: name: Griffith, Tia A
Publisher: name: Griffith University
Year: 2021
Subjects: subject: multimorbidity
subject: brain
subject: heart
subject: liver
subject: blood
subject: type 2 diabetes
subject: toll-like receptor
subject: Dysregulation

description: We live in an age of 'multimorbidity', yet have a rather poor understanding of the impacts of this phenomenon from cellular to organ and system levels. Bi-directional relationships are observed between cardiometabolic (diabetes mellitus, coronary heart disease) and mood disorders (major depressive disorder), each increasing disease risk and frequently co-existing. This cardiometabolic-mood interaction may reflect an under-appreciated commonality of underlying mechanisms across these pathologies. Importantly, comorbid development of these disorders exacerbates the already profound burdens of these individual chronic disorders. Further, psychosocial stress and ageing – increasingly prevalent features of modern societies - may promote these conditions and worsen outcomes. In assessing the intersections of these inter-related disorders, chronic low-grade inflammation is often highlighted and implicated as a common early substrate in cardiometabolic and mood disorders, linking these dominant diseases. Nonetheless, how immunoinflammatory dysregulation drives multimorbidity, and influences 'multifinality' (diverse disease outcomes from common risks/causes), are unclear. Whether immunoinflammatory changes in metabolic vs mood disorders are unique or involve common motifs, and how these interact in dictating cardiometabolic and mental health, remains to be established. Dysregulation of toll-like receptor (TLR) signalling has been specifically implicated in these diseases, potentially presenting a mechanistic nexus in comorbid development of cardiometabolic and mood disorders. The doctoral work presented in this thesis was designed to address the broad concept of mechanistic intersections contributing to rising multimorbidity. In order to practicably achieve this at a systems level, four major tissues impacted by and contributing to aspects of metabolic and mood disorders were studied: the brain, heart, liver and blood (circulating serum). The brain is an overarching and critical organ, regulating both mood and metabolic homeostasis, and influencing all other organ systems. Cardiac tissue is assessed as it is particularly impacted by type 2 diabetes (T2D); the risk of cardiovascular disease (CVD) is markedly elevated in T2D patients and is the lead cause of death in these patients. Further, CVD is an independent risk factor for both depression and T2D. The liver is fundamental to metabolic homeostasis (the central organ maintaining homeostasis across fed and fasted states via oxidisable substrate storage and release), links the gut to the periphery (gut-liver axis) and contains specialised macrophages which are front-line responders to gut-derived lipopolysaccharides (LPS) (arising with gut permeability changes). Finally, serum was assessed as it is the primary medium for transport of macronutrients, hormones and immunoinflammatory molecules throughout the body. To avoid the pitfalls of reliance upon gene-modified animal models, non-genetic murine models of type 1 and type 2 diabetes development in early adulthood were studied in Chapter 2 to explore potential changes in TLR-related signalling at the gene level. Somewhat unexpectedly, there was little evidence of shifts in TLR signalling in central nervous system (CNS), hepatic or cardiac tissues in these two conditions. Moreover, while modest elevations in circulating interleukin-6 (IL-6) are observed in T2D animals, tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) levels remained unchanged across models. Together with further analysis of gene expression, these data indicate that the T2D model does not develop an overtly pro-inflammatory phenotype, despite clear evidence of metabolic dysregulation. Given that a well-developed diabetic phenotype was observed (increased body weight, moderate hyperglycaemia, insulin resistance), this work called into question the essential role of TLR4 in the pathogenesis of T2D. Data does reveal marked changes in circulating adipokines and catecholamines, suggesting these endocrine systems may be key to disease progression, independent of inflammation. Surprisingly, assessment of circulating LPS revealed serum levels were decreased (although not significantly) in T2D animals, which casts doubt on whether 12 weeks of feeding with a ‘Western diet’ (30% of total calories from fat) is sufficient to disturb gut physiology and trigger the systemic innate immune response. The majority of animal models employ much higher fat percentages (45-60% calories from fat) in their diabetogenic diets, despite such high fat levels vastly exceeding those in a Western diet (~30%) and raising questions of pathophysiological relevance. Given that little evidence of TLR dysregulation was detected at a physiologically relevant fat percentage, a more chronic model (21-week dietary modification) with an intermediate fat content (43% calories from fat) was developed for subsequent experimentation. Studies in Chapter 3 demonstrate that a more profound weight gain is achieved with a higher fat content, which still remains below the extreme 60% levels often studied in animal models: this new feeding regime resulted in significantly greater body weight gain although fasting insulin and glucose levels were not altered when compared with the T2D model assessed in Chapter 2. Nonetheless, despite this more pronounced phenotype, limited TLR4-related gene changes were observed in the model. Selective changes to total cellular nuclear factor-kappa B (NF-κB) protein expression were observed in hepatic tissue, although analysis of the nuclear compartment revealed no changes in response to T2D. Thus, while a greater pool of this critical pro-inflammatory factor was evident, this was not necessarily associated with increased nuclear interaction and thus transcriptional control. These data suggest dysregulation of transcriptional control by NF-κB (particularly TLR4 pathway elements) may not be essential in the evolution of T2D in these animals. Examining a dietary intervention with an omega-3 polyunsaturated fatty acid (n-3 PUFA), α-linolenic acid (ALA), which has been linked to anti-inflammatory outcomes in some chronic disorders, revealed no improvements in the systemic sequalae of T2D. This is consistent with evidence PUFA supplementation may (somewhat paradoxically) have greater influences on metabolic homeostasis in healthy rather than diseased subjects. Unexpected elevations in key inflammatory transcripts were noted with ALA supplementation, potentially reflecting the highly pleiotropic actions of PUFAs. Proteomic profiling of cardiac tissue revealed that a number of inflammatory and related factors beyond TLR-related signalling were impacted by T2D (Serpin-1/PAI-1, leptin, resistin) and/or are sensitive to ALA (IL-10, CD40, VEGF). Data also suggest animal age may be a complicating factor in the protracted disease model, producing apparent independent effects on leptin/insulin expression levels, in turn complicating interpretation of these data. Finally, specific investigation of CNS changes in the model - via analysis of frontal cortex (FC) leptin receptor expression - indicates a sensitivity of central leptin signalling to T2D, which may not only participate in metabolic dysregulation but behavioural outcomes (as suggested in the pathogenesis of depression, for example). This highlights the likely importance of central control and behavioural determinants of disease outcomes. Investigating central control mechanisms and behaviour in more detail, studies presented in Chapter 4 revealed that the T2D phenotype involves induction of anxiety-related behaviours without impacting on hedonic behaviour. Further, despite evidence from other studies of the benefits of PUFA supplementation in mood disorders, ALA supplementation did not reverse anxiety-related behaviours, though increased locomotion in both healthy and T2D animals. Interestingly, analysis suggests ALA supplementation may confer benefits to locomotive activity independently of disease state, although outcomes are better in healthy controls. Examining elements of central reward pathways, neurotransmission, endocrine and inflammatory control in FC and hippocampus revealed select changes with T2D, including elevated Drd2 (dopamine D2 receptor) and reduced Htr1a (serotonin receptor 1A) in the FC, together with shifts in leptin receptor expression. Surprisingly, hippocampal Il1b gene expression remained similar between groups, though this is consistent with no change in hedonic behaviour. Circulating dopamine and leptin were also sensitive to T2D, with hippocampal dopamine levels selectively elevated in T2D animals supplemented with ALA (although the relevance of this finding is not clear). Overall, these data point to dysregulation of central dopamine and leptin signalling, which may contribute to behavioural disruption in T2D, in turn influencing disease development and outcomes. In the final studies, CNS responses were explored in greater detail via RNA-sequencing (Chapter 5), more broadly testing whether metabolic and mood disorders share common nervous system changes. Analyses of transcriptome profiles in the FC of the T2D model assessed in Chapter 5 together with a model of chronic social stress (SS) known to induce anxiety/depressive behaviours again provided limited support for an overarching immunoinflammatory dysregulation as a key driver of behavioural changes. These analyses reveal both distinct and common processes (beyond TLR-signalling) that are dysregulated in disease. Comparison between T2D and SS models reveal commonalities in CNS leptin and insulin receptor changes, congruent with evidence of insulin and leptin resistance in the T2D model and their implication in both human major depressive disorder (MDD) and T2D. A majority of over-represented genes were related to cell/tissue development, cell migration and proliferation, suggesting a dominant role for CNS 'remodelling' with both metabolic and mood disorders. Interestingly, down-regulation of ATP metabolic processes and mitochondrial genes was evident in the SS model but not T2D, supporting a more dominant effect of stress on energy production. Taken together, the findings presented in this doctoral project raise questions regarding the role of dysregulated TLR-signalling in T2D (and stress-related disorders). Despite a clear diabetic phenotype and shifts in behavioural profiles of T2D animals, findings challenge whether TLR-related dysregulation (reported by others) may be a consequence rather than causative factor in disease pathogenesis. Importantly, evidence is presented that insulin, leptin and dopamine signalling (together with other metabolic mediators) may be important linkages between metabolic and mood disorders, and underlie behavioural detriment in T2D. In terms of limiting the development or impacts of disease, n-3 PUFA supplementation resulted in selective benefits in T2D, though these appear unrelated to the metabolic profile of these animals. Finally, shared and unique CNS changes were identified in models of T2D and chronic social stress, supporting structural sensitivity and plasticity with inter-related metabolic and mood disorders.