Evidences from rodent studies have demonstrated that AICAR stimulates fatty acid uptake and fatty acid oxidation in muscle [10, 18, 19], heart  and liver . The stimulation of fatty acid oxidation is recognized as the consequence of phosphorylating and inhibiting ACC, subsequently reducing the concentration of malonyl-CoA, the enzymatic product of ACC and the physiological inhibitor of CPT I. The reduction of malonyl-CoA concentration reduces CPT I inhibition, and thereby increases the fatty acid oxidation. Indeed, in adult animals it is well established that fatty acid oxidation is controlled mainly by the variation of malonyl-CoA concentration and the sensitivity of CPT I to malonyl-CoA inhibition in liver under many physiological conditions. In adult rat hepatocytes, reduction of malonyl-CoA concentration by glucagon significantly increases fatty acid oxidation. However, in this study the concentration of AICAR adapted from rodent species was sufficient to change malonyl-CoA concentrations in rat or mice, but there was no effect on the total [1-14C] palmitic acid oxidation in hepatocytes isolated from suckled neonatal piglets. In agreement with the fatty acid oxidation rate, the malonyl-CoA sensitive CPT activity and inhibition of its activity by malonyl-CoA assayed in cell homogenates remained unchanged among the treatments. The dampened responses of fatty acid oxidation to AICAR treatment could be associated with the species differences and the specific physiological status of the hepatocyte at the time of isolation. First, low lipogenesis and limited fatty acid oxidation capacity are observed in hepatocytes isolated from neonatal swine. Results from earlier studies demonstrated that the rate of lipogenesis is very low in isolated hepatocytes from both fed and fasted newborn pigs , suggesting that malonyl-CoA concentration could be negligible during early neonatal life. Meanwhile, the oleate oxidation and ketogenesis is about 70 and 80% lower in mitochondria isolated from newborn piglets than adult rats , and more than 90% of the oleate taken up by the hepatocyte converts to esterified fat , suggesting that newborn piglets have a low fatty acid oxidative capacity. However, the extremely low fatty acid oxidation is apparently not due to the CPT I inhibition, because the lipogenesis and malonyl-CoA concentration measured in hepatocytes isolated from newborn piglets is very low [1, 22]. Therefore, the attenuated response to AICAR might be due to a low baseline concentration malonyl-CoA in the neonatal piglets hepatocytes. Secondly, evidence from literature indicates that the regulation of fatty acid oxidation during the neonatal period is different from adult animals. It is likely that the control of fatty acid oxidation is primarily effected by variation in sensitivity of CPT I to malonyl-CoA inhibition rather than by a change in malonyl-CoA concentration . Indeed, we found that the considerable increase of fatty acid oxidation in hepatic mitochondria isolated from 24 h-old piglets was paralleled with a significantly decrease in sensitivity of CPT I to malonyl-CoA inhibition . Moreover, the decrease in sensitivity of CPT I to malonyl-CoA inhibition was related to the food intake, because the IC50 obtained from 24 h-old fed piglets much higher than that from 24 h-old fasted and newborn piglets . Similar results were also observed in our previous studied using hepatocytes and liver homogenate [2, 24]. Because the hepatocytes isolated in this study were from 32 h-old fed piglets, the reduced response to AICAR might also be due an increased IC50 after the piglets suckled. Similar results were observed in muscle isolated from fasted rats , suggesting that the stimulation of fatty acid oxidation by AICAR depends on nutritional status. Thus, the stimulation of fatty acid oxidation by AICAR might be limited by the age-related physiological status.
Although AICAR did not change the total fatty acid oxidation, addition of AICAR to the cells decreased CO2 production by 18%, resulting in a significant difference in distribution of oxidative products between CO2 and ASP compared to the control. Consistent with the distribution change, we found that addition of AICAR increased ACC activity in hepatocytes, and the increase was promoted by adding insulin to the cells treated with AICAR. Inclusion of citrate in incubation medium also stimulated ACC activity in the cells, but the stimulation was higher in control cells than in cells treated with AICAR. These results suggest that the increased ACC activity induced by AICAR might drive the end product of beta-oxidation, acetyl-CoA, toward fatty acid synthesis, resulting in a decrease of CO2 production from fatty acid oxidation. As already discussed, the nutritional and physiological status of the isolated hepatocytes might be associated with the abrogated response of fatty acid oxidation to AICAR, but we have not evaluated the malonyl-CoA concentrations. If AICAR increases ACC activity, the malonyl-CoA concentration would be increased in the cells. It appeared that the increase of malonyl-CoA did not lead to a change in CPT I activity, the result might imply that the increases did not reach the inhibition level required by the CPT I in the cells with a high IC50 value due to the fed status. Even so, the phenomenon of increasing ACC activity could not be fully explained. Both isomers of ACCα and ACCβ are expressed in the liver, and ACCα sustains the regulation of fatty acid synthesis while ACCβ mainly controls fatty acid oxidation. The assay performed in this study could not distinguish the activity of ACCα and ACCβ, but their expression can be regulated by promoters at the transcriptional level in which nutritional status can play an important role. In addition to regulation at transcriptional level, ACCα and ACCβ are regulated by phosphorylation and dephosphorylation at the metabolic level. The phosphorylation is due to an increase of AMP levels when the energy status of the cells is low, resulting in the activation of AMPK. The cell energy level was high in this study, but AICAR is an activator of AMPK and its activation is considered to be independent of energy status of the cells . Thus, the opposite influence of AICAR on ACC in newborn suckled pigs needs to be investigated further in both regulatory levels under the specific physiological and nutritional conditions. Particularly, the role of AMPK and insulin in regulation of ACC has not been studied and need to be examined in the neonatal pig. Further investigation is necessary for a better understanding of the energy and metabolic regulation mechanism in the newborn pigs. In summary, AICAR may affect the distribution of metabolic products from fatty acid oxidation in hepatocytes isolated from suckled neonatal pigs by changing ACC activity. The effect of AICAR on ACC activity will be impacted by citrate concentration in the cells.