C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity

Abstract

C75, a known inhibitor of fatty acid synthase is postulated to cause significant weight loss through decreased hypothalamic neuropeptide Y (NPY) production. Peripherally, C75, an alpha-methylene-gamma-butyrolactone, reduces adipose tissue and fatty liver, despite high levels of malonyl-CoA. To investigate this paradox, we studied the effect of C75 on fatty acid oxidation and energy production in diet-induced obese (DIO) mice and cellular models. Whole-animal calorimetry showed that C75-treated DIO mice had a 50% greater weight loss, and a 32.9% increased production of energy because of fatty acid oxidation, compared with paired-fed controls. Etomoxir, an inhibitor of carnitine O-palmitoyltransferase-1 (CPT-1), reversed the increased energy expenditure in DIO mice by inhibiting fatty acid oxidation. C75 treatment of rodent adipocytes and hepatocytes and human breast cancer cells increased fatty acid oxidation and ATP levels by increasing CPT-1 activity, even in the presence of elevated concentrations of malonyl-CoA. Studies in human cancer cells showed that C75 competed with malonyl-CoA, as measured by CPT-1 activity assays. Thus, C75 acts both centrally to reduce food intake and peripherally to increase fatty acid oxidation, leading to rapid and profound weight loss, loss of adipose mass, and resolution of fatty liver. The pharmacological stimulation of CPT-1 activity is a novel finding. The dual action of the C75 class of compounds as fatty acid synthase inhibitors and CPT-1 agonists has therapeutic implications in the treatment of obesity and type II diabetes.

Figure 1

C75 caused sustained weight loss and reduced food consumption in DIO mice. (A) Three mice were treated on day 0 with C75 (20 mg/kg, i.p.; ▴). Subsequent doses were administered as follows: 15 mg/kg, day 2; 10 mg/kg, day 4; 15 mg/kg, days 6 and 8 (arrows). Four mice received RPMI medium 1640 (○). Weight and food consumption were measured daily. After the initial 16% loss of body mass within the first 2 days of C75 treatment, weight loss stabilized 20–22% of initial body mass over the course of the experiment, whereas controls gained 1% (P < 0.0001 by unpaired t test). Error bars represent standard error of the mean. (B) C75 reduced food consumption in DIO mice. C75-treated mice (□) ate on average 1.83 ± 0.29 g/day compared with 2.72 ± 0.21 g/day for controls (▪).

Figure 2

C75-treated mice expended more energy than pair-fed controls by increasing fatty acid oxidation. (A) C75-treated mice maintained an average caloric expenditure of 0.79 kcal/h (red) compared with 0.53 kcal/h (black) in the pair-fed mice P < 0. 0001, unpaired t test, n = 2). (B) In contrast, the RER is essentially the same in both pair-fed (average = 0.753) and C75-treated (average = 0.758) mice, indicating fatty acid oxidation. The C75-treated mice lost 4.4% of body mass and consumed an average of 1.9 g of food per 20 h, whereas the pair-fed mice lost 2.0% of body mass (data not shown). (C) Etomoxir produced a dose-dependent decrease in energy production in C75-treated mice. After 24-h adaptation to the Oxymax chambers, four mice (black) received C75 [15 mg/kg, i.p.] at the time indicated by the arrow. Two hours after C75 treatment (arrow), two mice received etomoxir at the following doses (mg/kg, i.p.): 5 (orange), 10 (red), 15 (green), 20 (blue), and the other two received vehicle control (black). Each dose of etomoxir constituted a separate experiment. (D) Analysis of average heat production for each dose from the time of C75 treatment until the end of the experiment shows that etomoxir significantly reduced energy production in C75-treated mice for all concentrations tested (P < 0.0001, unpaired t test, four mice in each test group, 154 determinations for each group). Error bars represent the standard error of the mean (***, P < 0.001).

Figure 3

C75 increased fatty acid oxidation, ATP production, and CPT-1 activity in mouse 3T3-L1 adipocytes. (A) C75 caused a concentration-dependent increase in fatty acid oxidation. At doses of 30 and 40 μg/ml, C75 significantly increased fatty acid oxidation by 203% (P < 0.02) and 358% (P < 0.003, unpaired t test, n = 5), respectively. (B) ATP levels also showed a concentration-dependent increase after C75 treatment. C75 treatment significantly elevated ATP at 20 μg/ml (P = 0.018) and 30 μg/ml (P = 0.002, unpaired t test, n = 7). (C) C75 increased CPT-1 activity. Malonyl-CoA at 50 μM inhibited CPT-1 activity to 41% (P = 0.02, unpaired t test, n = 3) of control, whereas C75 increased CPT-1 activity to 213% (P < 0.0001, unpaired t test, n = 3) of control. CPT-1 activity was normalized to cell number as nmol of palmitoylcarnitine synthesized per 10⁵ cells per min. Error bars represent standard error of the mean (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4

C75 increased fatty acid oxidation and CPT-1 activity in primary rat hepatocytes. (Left) C75 treatment caused a dose-dependent increase in fatty acid oxidation up to 839% of control at 40 μg/ml (P < 0.0001, unpaired t test, n = 3). (Right) C75 treatment increased CPT-1 activity up to 475% of control at 30 μg/ml (P < 0.0001, unpaired t test, n = 3). CPT-1 activity was normalized to cell number as nmol of palmitoylcarnitine synthesized per10⁶ cells per min. Error bars represent standard error of the mean (***, P < 0.001).

Figure 5

C75 increased CPT-1 activity in the presence of inhibitory concentrations of malonyl-CoA and is a competitive CPT-1 agonist. (A) C75 increased CPT-1 activity up to 166% of control at 20 μg/ml (P = 0.0027, unpaired t test), whereas malonyl-CoA (20 μM) alone reduced activity to 64% of control (P = 0.0029, unpaired t test, n = 3). There was no significant difference in CPT-1 activity between the C75 or C75 + malonyl-CoA treated cells (unpaired t tests, n = 3). (B) Malonyl-CoA in the assay buffer (left bar) reduced CPT-1 activity by approximately 70%. For the middle bar, cells were pretreated for 2 h with C75, but C75 was not present in the assay buffer. Nonetheless, C75 pretreatment increased CPT-1 activity to 158% of control (P = 0.0004, unpaired t test, n = 3). When cells were pretreated with C75 for 2 h and C75 was removed from the assay buffer and replaced with malonyl-CoA right bar, CPT-1 activity fell to approximately 50% of control (P = 0.001, unpaired t test, n = 3). CPT-1 activity was normalized to cell number as nmol of palmitoylcarnitine synthesized per10⁶ cells per min. Error bars represent standard error of the mean (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 6

Proposed model for central and peripheral C75 mechanisms of action. Centrally, C75 alters orexigenic and anorexigenic peptides, leading to a net reduction in food consumption. In the peripheral organs such as liver and adipose tissue, C75 increases CPT-1 activity, leading to increased fatty acid oxidation and energy production. Both mechanisms contribute to weight loss, but the peripheral mechanism is responsible for the selective reduction in adipose tissue mass and resolution of fatty liver.