Effects of c75 on Fatty Acid Synthesis and Fatty Acid Oxidation

C75 mechanism of action involves the main components of fatty acid metabolism. The proposed action of C75 is that it activates AMP-activated protein kinase (AMPK) which then inactivates pathways that are involved in fat synthesis and activates pathways involved in fat degradation. AMPK is an energy sensor molecule that becomes active when energy levels are low. C75 activates AMPK when energy levels are not low and so C75 tricks AMPK into signaling a low-energy state.The end result is less energy storage and more energy expenditure.

Acetyl-CoA is converted to malonyl-CoA by an ATP-dependant acetyl-CoA carboxylase (ACC). Diagram 2 shows this reaction schematically. This reaction is the rate-limiting step for long chain fatty acid synthesis. C75 decreases ACC activity as shown in Figure 3. Western blot analysis showed that the amount of phosphorylated, or active, ACC decreased with the administration of C75. Because the carboxylation of acetyl-CoA by ACC requires ATP, cellular ATP levels were measured after C75 administration. It is probable that these changes in ATP cause the stimulation of AMPK. Initially, 2.5 minutes after C75 administration, ATP levels drop by about 65%. This sudden drop in energy level activates downstream pathways. This initial decease in ATP is cause by futile cycling of ACC, since FAS is inhibited. Malonyl-CoA is synthesized, and then likely converted back to acetyl-CoA by malonyl-CoA decarboxylase. After about two hours the ATP levels rise to about 268% of the control (Figure 5.) The fluctuations of ATP levels affect AMPK activity.

Fatty Acid Synthase (FAS) is a multi enzyme complex that generates long chain fatty acids. Malonyl-CoA and acetyl-CoA are the substrates. C75 is an inhibitor of FAS by preventing malonyl-CoA from binding to the ß-ketoacyl synthase domain. FAS is inhibited by c75 in primary cortical neurons. The IC 50 ,which is the concentration of c75 that inhibits FAS by 50%, was found to be 40µg/ml (figure 2A landree). The IC50 is the concentration used in all other experiments mentioned.

C75 up-regulates fatty acid oxidation by activating Carnitine palmitoyltransferase-1 (CPT-1). CPT-1 is an integral mitochondrion protein that catalyzes esterification of long chain Acyl-CoA to L-caritine. This reaction is the rate determining step in the transport of acyl compounds from the cytosol to the mitochondrion where they undergo further oxidation. C75 stimulates CPT-1 by about 217% (fig 2B landree) and increases fatty acid oxidation by 150% (after two hours, fig 2C). The result is an increased energy level and subsequent neuronal activation (figure). Malonyl-CoA is an inhibitor of CPT-1 so it has an .

AMP-activated protein kinase (AMPK) altered neuronal energy level affects appetite. AMPK is involved in whole body energy balance because it acts as an intracellular energy sensor. AMPK activity is decreased by C75. AMPK has to be phosporylated and bound to AMP to be active. Western blot analysis revealed that the amount of phosphorylated AMPK decreases with the presence of C75. The long term increase of ATP can be attributed to AMPK because it activates glycolysis. AMPK activates ACC by phosphorylating it.

The mechanism of C75 involves the main enzymes of fatty acid metabolism and fluctuations of ATP levels. By inhibiting FAS it shortly depletes ATP which then activates a signaling pathway with AMPK. It is apparent that the regulation comes from these fluctuations of high energy compounds and fatty acid metabolism intermediates. The regulation of appetite is not regulated by a single enzyme or pathway, but by the flux of several pathways and their products. C75 has further confirmed that such regulation exists and is central to whole-body energy regulation.

                           Diagram 4. The effects of C75 on fatty acid                                     metabolism in the hypothalamus. (Landree 2004)

Diagram 2. Acetyl-CoA Corboxylase reaction.

Figure 3. Western blot shows the decreased amount of phosphorylated ACC in the presence of C75. ACC needs to be phosphorylated to be active. (Landree 2004)

Figure 4. Cellular ATP levels experience a sudden drop and then a slow rise after C75 treatment. (Landree 2004)

Figure 5. Long term ATP levels increase with C75 treatment. (Landree 2004)

Diagram 3. FAS reaction.

Figure 6. C75 inhibits FAS. The incorporation of radio labeled acetic acid decreases as the amount of c75 increases. (Landree 2004)

Figure 7. CPT-1 activity increases with C75 after 2 hr. treatment with C75.(Landree 2004)

Figure 8. Increased fatty acid oxidation was seen with C75 treatment. The total oxidation of labeled palmitate was calculated by measuring the sum of labeled carbon dioxide and labeled soluble products during a 30 min. incubation.(Landree 2004)

Figure 9. Decreased amount of phosphorylated AMPK with C75 treatment. (Landree 2004)

Figure 10. ATP levels and AMPK activity with C75 treatment. (Landree 2004)

Figure 11. With C75 treatment AMPK alters the rate of glycolysis. (Landree 2004)