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  • In order to elucidate the mechanisms behind the observed

    2021-09-24

    In order to elucidate the mechanisms behind the observed changes in metabolic activity in the cardiomyoblasts we investigated changes in gene and protein expression subsequent to treatment with PSN632408 with or without concurrent exposure to palmitate. Activating GPR119 in basal and high palmitate conditions led to distinct changes in gene and protein expression. This is of interest and confirmed that alterations in lipid status, which is a hallmark of metabolic disease, can modulate the signalling pathways through which GPR119 elicits its effects. Specifically, exposure to palmitate largely abrogates the effects of GPR119, particularly blunting its effects on mRNA expression of genes that mediate metabolic and hypertrophic pathways. This abrogated effect in the presence of metabolic dysregulation is further consistent with findings of GPR119 agonists in skeletal muscle myotubes and in early clinical data in which various GPR119 agonists did not improve glycaemia in male subjects with type 2 diabetes (Cornall et al, 2013b, Katz et al, 2012, Nunez et al, 2014). The fact that in the absence of any palmitate exposure, PSN632408 decreased the abundance of a number of mRNAs coding for the master regulators of energy metabolism including AMPKα, NFATc3 and PPARα is consistent with decreased metabolic capacity as these genes act to promote both glucose and fatty carnosic acid synthesis oxidation. The heart has an unremitting continuous requirement for ATP based on its inherent workload and the adult heart relies predominantly on fatty acids as substrate for ATP (Kolwicz and Tian, 2009). Thus this profile of mRNA expression subsequent to GPR119 agonism is likely to impinge on overall cardiac muscle energetics and in turn reduce cardiac efficiency and function. In conditions of high palmitate exposure there was an additive reduction in metabolic carnosic acid synthesis activity compared to palmitate treatment alone, suggesting a worsening of metabolic function. However this occurred in the absence of changes in oxidative gene expression. In fact the only change in metabolic gene expression was an increase in SOCS3 abundance in cells continually exposed to palmitate for the entire treatment period which is opposite to the reduction in SOCS3 expression which was observed in cells without palmitate treatment. SOCS3 is known to inhibit insulin signalling in the heart (Calegari et al., 2005). Thus in normal cells the observed reduction may enhance insulin signalling whereas in the presence of palmitate treatment insulin signalling might be further impaired by increased SOCS3 expression. In addition to this, phosphorylated JNK protein was also increased (implying increased activity) in cells which were temporarily exposed to palmitate. JNK has previously been shown to induce serine phosphorylation of the insulin receptor substrate-1 to impair insulin signalling/sensitivity (Aguirre et al, 2000, Kang et al, 2011). Thus increased JNK phosphorylation in this condition could translate to reduce insulin sensitivity and impaired glucose metabolism. This is of concern given that palmitate is known to decrease glucose uptake in cardiac muscle cells and indeed that the heart becomes insulin resistant in type 2 diabetes further limiting substrate availability (Abel et al, 2012, Iozzo et al, 2002, Nobuhara et al, 2013, Wang et al, 2009, Wei et al, 2013). Thus, it would be hoped that anti-diabetic agents would improve cardiac muscle insulin sensitivity. Interestingly, in the 22 hour timeframe of the current study there was no effect of GPR119 agonism on lipid accumulation in cells treated with palmitate. However long-term, if there are impairments on fatty acid oxidation as the gene expression profile implies, intramyocellular lipid accretion is likely to ensue. Given the observed divergence in gene and protein expression, it is wholly possible that GPR119 exerts its effects in healthy metabolically and deregulated states via distinct signalling pathways. However, either way it appears the effects of GPR119 activation on metabolism in the heart are unlikely to be beneficial and in the long term may increase the risk of cardiomyopathies.