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Title: The cyclic AMP signalling system as a regulator of preadipocyte differentiation
Author: Yarwood, Stephen J.
ISNI:       0000 0001 2447 4655
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1997
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A study was carried out on the fibroblastic 3T3-F442A preadipocyte cell system to investigate the role of the adenylate cyclase signalling system in modulating their adipocyte differentiation. Three areas were investigated: 1) The expression of heterotrimeric guanine nucleotide binding protein (G-protein) subunits during individual stages of cellular differentiation. 2) The role of cyclic AMP in modulating preadipocyte differentiation. 3) Interactions occurring between the cyclic AMP and MAP kinase signalling cascades during the differentiation process. The differentiation of 31.3-F442A preadipocytes was shown, under serum-free conditions, to be divided into at least two stages. The first stage is dependent on the presence of growth hormone (GH) which primes cells to the action of other differentiative agents. The second, maturation, stage involved a combination of insulin, EGF and T3 which acted on GH-primed cells to promote terminal differentiation. Terminal differentiation was determined by two criteria; morphologically by the accumulation of Oil Red O-positive triacylglycerides in the cell cytoplasm and biochemically, by the emergence of the specific activity of an adipocyte-specific enzyme marker, alpha-glycerophosphate dehydrogenase. A range of specific anti-peptide antisera were generated and used to quantify specific changes in the expression of stimulatory- and inhibitory-G-proteins during the two-stages of 3T3-F442A preadipocyte differentiation. Stimulatory-G-proteins (GS) couple adenylate cyclase to positive-acting extracellular stimuli, whereas inhibitory-G-proteins (GO mediate the actions of negative-acting stimuli. Undifferentiated 3T3-F442A preadipocytes were found to express detectable levels of the GS alpha-subunits, Gsalpha42, Gsalpha44, and the Gi alpha-subunits, Gi2alpha and Gi3alpha in their cell membranes, together with the 36 kDa beta-suhunit (Gbeta36) which is subunit common to both GS and Gi. During the GH-priming stage of adipocyte differentiation significant increases in protein expression were observed for GSalpha42, GSalpha44. Gi2alpha and Gbeta36, but not Gi3alpha. Increased levels of GSalpha44 and Gbeta36 were sustained during the first two days of maturation and then fell, in terminally differentiated cells, to levels observed in undifferentiated 3T3-F442A fibroblasts. Levels of Gi2alpha were also sustained during the initial stages of maturation, but then fell, together with Gi3alpha, to levels significantly lower then those found in undifferentiated fibroblasts. In contrast, levels of GSalpha44 were maintained at a significantly elevated level in terminally differentiated cells when compared to undifferentiated fibroblasts. Changes in GSalpha subunit expression were also observed in non-differentiating control cultures, in the absence of differentiative agents. This G-protein may play a role in modulating preadipocyte differentiation. Indeed, treatment of cell with cholera toxin, which constitutively activates GS and adenylate cyclase, dramatically inhibited differentiation of 3T3-F442A cells by ~90%. Adenylate cyclase catalyses the formation of the intracellular second messenger cyclic AMP which then hinds to and activates protein kinase A. Direct activation of adenylate cyclase with forskolin (50muM) or treatment with the cyclic AMP analogue CPT-cyclic AMP (0.25mM) was found to potently inhibit the adipose conversion of 3T3-F442A preadipocytes induced with foetal calf serum and insulin (FCS/insulin; ~90% inhibition) or with a serum-free hormonally defined medium (DDM; ~70% inhibition). In contrast, treatment of cells with the cyclic AMP phosphodiesterase inhibitor, IBMX, or with low concentrations of CPT-cyclic AMP (10nM) or forskolin (10nM) potentiated cellular differentiation induced with FCS/insulin (~80-99% increase) or DDM (~30-40% increase). Both IBMX and 10nM forskolin induced small and relatively transient increases in intracellular cyclic AMP (~8 and ~3 fold maximal increase), whereas those induced by 50muM forskolin were much larger and more prolonged (~120 fold maximal increase). This suggests that the differential effects of cyclic AMP on the adipose conversion of 3T3-F442A cells could he attributable to interactions occurring at different stages of the differentiation program. Indeed, inclusion of 10nM forskolin or IBMX during the GH-priming stage of differentiation synergistically enhanced GH-promoted differentiation (~90% and ~130% increase, respectively). In contrast, when included during the maturation stage, these agents were found to inhibit terminal differentiation (~60% and ~70% inhibition, respectively). A potential mechanism underlying the effects of cyclic AMP on cell growth is through functional interplay with the MAP kinase signalling cascade. MAP kinases were potently activated in 3T3-F442A preadipocytes by the differentiative factors GH (~5 fold activation), insulin (~5 fold activation) and EGF (~20 fold activation). Antisense depletion of MAP kinase was found to severely retard the differentiation of 3T3-F442A cells with serum or DDM by approximately 95%, demonstrating that adipose conversion of these cells displays an overall requirement for MAP kinases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Lipid metabolism; Cell culture