Sustained over time, the lower ATP yield could signal a down-regulation of lipogenesis at the level of pathway flux, enzyme synthesis, or both. Down-regulation of lipogenic flux could involve a change in cellular redox state. Long-term treatment with FCCP significantly increased the NAD+ to NADH ratio. Forced UCP1 expression elicited a similar trend, although the ratio change was not statistically significant. The NAD+ to NADH ratio has been linked to the activities of several metabolic signaling pathway components, including Sirt1, Clock, and CtBP. Another possible regulatory response is that the lower ATP yield Digoxin stimulated the AMP-activated protein kinase pathway. In adipocytes, AMPK serves as a metabolic master switch. Once stimulated, the AMPK pathway can reduce ATP utilization by inactivating enzymes involved in biosynthesis, including lipogenesis. Elevated AMPK activity has been found in white adipose tissue of ap2-UCP1 transfected mice, adenovirus-induced hyperleptinemic rats, adiponectin treated rats, and rats after exercise. Interestingly, these animal studies also found reduced TG levels in the adipose tissue. The AMPK pathway has been shown to stimulate glucose transport and glycolysis, which could explain the up-regulation of glucose uptake and lactate output observed in the present study. On the other hand, AMPK activation by mitochondrial Licochalcone-B uncoupling was not confirmed in the present study. Establishing a direct role for this and other signaling pathways warrants further studies involving knock down or inhibition experiments. While the qualitative effects of UCP1 and FCCP were substantially similar, there were quantitative differences. In general, the chemical uncoupler generally elicited larger responses. For example, FCCP treatment increased the OUR by 55%, but UCP1 expression did not result in a significant change. Long-term treatment with FCCP significantly depressed the cellular ATP level even in the presence of high glucose in the culture medium. Forced expression of UCP1 did not significantly depress the ATP level relative to pRev control unless glucose was removed from the medium.