Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Subject "Acute phase response -- Synthesis"

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    Examining plasminogen activator-inhibitor-1 and C-reactive protein regulation by glucocorticoids and pro-inflammatory cytokines in liver cell lines
    (Stellenbosch : Stellenbosch University, 2021-12) Dale, Lieke; Verhoog, Nicolette J. D.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Current knowledge suggests that a continued elevated acute phase response (APR), which is often referred to as low-grade inflammation, occurs during insulin resistance. The levels of acute phase proteins (APPs), such as plasminogen activator-inhibitor-1 (PAI-1), C-reactive protein (CRP), and serum amyloid A (SAA) are significantly increased during this response, hence why they are commonly used as biological markers for type 2 diabetes (T2D). In the liver, APPs are regulated by the peripheral mediators of stress (i.e., endogenous glucocorticoids (GCs)) and inflammation (i.e., pro-inflammatory cytokines), with both implicated in the development of insulin resistance. Exogenous GCs are routinely prescribed in the management of inflammatory disorders. However, long-term GC treatment can exaggerate insulin resistance as it could possibly disrupt the interplay between endogenous GCs and cytokines, leading to a recurrent, chronic APR. Current knowledge suggests that when it comes to the APR, GCs, which are traditionally known for its anti-inflammatory properties, exert more pro- inflammatory behaviour, by further strengthening cytokine-induced APP expression. Whilst this cooperative regulation by GCs and pro-inflammatory cytokines have been extensively studied and demonstrated for SAA, fewer have studied this phenomenon in regards to PAI-1 and CRP, especially in the liver. In addition, previous studies investigating the possible co- regulation of PAI-1 and CRP by GCs and pro-inflammatory cytokines, included only interleukin-6 (IL-6) or tumour necrosis factor-alpha (TNF-α) as representative pro- inflammatory cytokine, respectively. Therefore, the current study examined the expression of both PAI-1 and CRP in response to GCs (synthetic and endogenous) in the presence of either pro-inflammatory cytokine, TNF-α or IL-6, in a murine (BWTG3)- and human (HepG2) liver cell line. The effects were investigated at both the mRNA- and intracellular protein level, using real-time semi-quantitative polymerase chain reaction (qPCR) and western blotting, respectively. In addition, the potential underlying molecular mechanism governing the action of these biological mediators to affect APP expression were investigated, using a luciferase promotor-reporter assay. PAI-1 and CRP levels generally remained elevated in response to individual and combinatorial treatments with GCs and pro-inflammatory cytokines, at intracellular protein- and promotor level. However, only under certain conditions, cooperativity was displayed between the test compounds, where PAI-1 and CRP expression were further potentiated, more than what was observed with either test compound alone. APPs are secreted proteins; therefore, the rate of protein synthesis and export presumably plays a role in determining intracellular APP expression and highlights the importance of measuring both intra- and extracellular APP expression, the latter of which the current study did not determine. In addition, the increase in PAI-1 and CRP promoter activity does suggest that the molecular mechanism of action whereby GCs and pro-inflammatory cytokines increase PAI-1 and CRP levels is at the promoter level. Whether only the proximal promotor is involved remains to be elucidated. Taken together, the findings of the current study give credence to the current knowledge available on the subject of cooperative regulation of APPs by GCs and pro- inflammatory cytokines. It is clear that GCs favour an increase in APP expression, at times enhancing the cytokine-induced PAI-1 and CRP expression. However, GCs were also able to antagonise the cytokine-induced PAI-1 and CRP expression, which is in line with their anti- inflammatory role. Ultimately, our findings highlight the diversity and complexity to the often- contradictory nature of GCs and their crosstalk with inflammatory mediators. Keywords: acute phase proteins, acute phase response, cooperative regulation, C-reactive protein, glucocorticoids, hepatic insulin resistance, inflammation, plasminogen activator- inhibitor-1, pro-inflammatory cytokines, stress