The glucagon stimulation test was performed in fasted rats and no significant difference was observed between fasted TGR and SD rats. However, this result can be attributed to the action of glucagon in all the metabolic sensitive tissues of the rat (such as muscle) and not exclusively in the liver. Glycogenolysis was evaluated through baseline hepatic glycogen concentration and levels of hepatic glycogen phosphorylase, an allosteric enzyme responsible for catalyzing the phosphorylation of glycogen to glucose1-P, playing a fundamental role in glycogen SP600125 chemical structure metabolism [7] and [26]. There was no significant

difference in hepatic glycogen phosphorylase levels analyzed by Western blotting. The absence of alteration in glycogenolysis pathway can explain the unaltered hepatic glycogen levels in TGR. To evaluate gluconeogenesis pathway separately we performed the pyruvate challenge test [18]. The first regulated step in the gluconeogenic pathway from pyruvate and its precursors is the pyruvate to oxaloacetate carboxylation, catalyzed by ATP-dependent pyruvate carboxylase [8], [9] and [10]. The pyruvate challenge experiment showed

that overnight fasted TGR rats have a decrease in buy AZD2281 the glucose synthesis when compared to overnight fasted SD rats, suggesting a downregulation in the gluconeogenesis pathway, since overnight fasted rats have negligible amounts of preformed glycogen. In order to confirm the downregulation of the gluconeogenesis pathway, it was evaluated the mRNA expression of the key enzymes of this route. The expression of G6Pase, a multicomponent enzyme system that hydrolyses glucose-6-phospate (G6P) to glucose in the final step of gluconeogenesis, showed no statistically difference in TGR and SD rats. PEPCK, one of the main rate-limiting enzymes of gluconeogenesis, simultaneously decarboxilates and phosphorylates oxaloacetate to phosphoenolpyruvate, had its expression significantly reduced in TGR when compared to SD rats. These results suggest that the gluconeogenesis downregulation could be due to the decreased expression of PEPCK. Recently, it has been documented that HNF4α has been implicated in gluconeogenesis through transcriptional

regulation of G6Pase and PEPCK, which are before rate-limiting enzymes in this process as discussed previously [27]. The mRNA expression of HNF4α analysis by RT-PCR showed significantly decreased levels in TGR, when compared to SD rats. This finding pointed out to a relation between Ang-(1-7) and HNF4α, leading to an overall downregulation of gluconeogenesis. This result can be responsible, at least in part, for the improved circulating glycemic profile in TGR described previously [23]. In summary, the results obtained in the present study show that transgenic rats with increased Ang-(1-7) plasma levels, present a lower activation of the gluconeogenesis pathway responsible for glucose synthesis, without evidence of alteration in the hepatic glycogenolysis.