Small molecules that inhibit mitochondrial respiration have been indispensable for investigating mitochondrial function and regulation, and new modulators offer possibilities for uncovering mechanisms which are as yet undiscovered. In our screen of 32 natural compounds, Ppc-1 was especially interesting as a compound with oxygen consumption-stimulating activity but without inhibitory effect on ‘state 3’. ‘State 3’ is induced when both respiratory substrates and ADP are supplied to mitochondria. The electron transfer system and ATP synthase are strictly regulated, through molecular interactions and intra-mitochondrial signaling systems which under various conditions can be labile, reducing ATP synthesis and producing excess reactive oxygen species [ 27 – 30 ]. Ppc-1 at >98% purity enhances oxygen consumption rate in a dose-dependent manner ( Fig. 2E ), showing linear correlation with the RCR for ADP ( Fig. 2E ). However, Ppc-1 does not serve as a respiration substrate itself, and its oxygen consumption kinetics show that it does not affect ATP synthase ( Fig. 2F ). These results suggest that Ppc-1 is a novel mitochondrial uncoupler.

Ppc-1 induces weight loss

We evaluated the effects of Ppc-1 on weight and fat content by administering it directly to mice. We found that Ppc-1 significantly suppresses weight gain in mice. It is noteworthy that this effect of Ppc-1 is observed at 21°C, a typical vivarium temperature that is below thermoneutrality [31]. Ppc-1 also leads to higher serum amounts of fatty acids and triacylglycerols, with lower percentages of subcutaneous and visceral fat compared to control mice. However, no other serum parameters show significant differences between Ppc-1-treated and control mice. The serum levels of ALT, BUN and adiponectin are all normal, and we observed no abnormalities in liver and kidney tissues. We initially guessed that Ppc-1 might induce adverse effects such as growth inhibition, since Ppc-1 inhibited growth of cultured cells [19] (S3 Fig.). However, we found that Ppc-1 concentrations were very low in serum and in all tissues tested in mice that were administered the compound (Fig. 7). Furthermore, no ectopic fat was detected in Ppc-1-treated mice. Thus, we speculate that Ppc-1 functions as an uncoupler in adipocytes and other cells in Ppc-1-treated animals, reducing the efficiency of mitochondrial ATP production. No excess heat production was observed, possibly because the Ppc-1 absorption and uncoupling reaction proceeded slowly over the course of serial administration. Low concentrations of Ppc-1 in the liver and kidney suggest a rapid degradation or modification by metabolic pathways. On the other hand, it is interesting that Ppc-1 stimulates adipocyte-like 3T3-L1 cells to release fatty acids into the culture medium (Fig. 7). Although the mechanism underlying the fatty acid release is under investigation, these findings are likely to be closely related to the increased serum fatty acids observed in mice treated with Ppc-1.

Obesity is a complex multifactorial disease and is a risk factor for other chronic diseases, including type 2 diabetes and cardiovascular disease. Understanding the metabolic factors and mitochondria dysfunction associated with obesity and weight loss success is important for developing appropriate prevention and treatment strategies [32]. Drugs for weight loss have been sought by many studies. DNP is reported to cause rapid weight loss, but leads to an unacceptably high number of adverse side effects [33–35]. Amphetamine derivatives have been used as appetite suppressants, but induce side effects that include valvular heart disease and lung hypertension. A new centrally active appetite suppressant and a lipase inhibitor are currently being used as diet pills, but have equally unpleasant side effects. A small molecule, AdipoRon, which binds to adiponectin receptors, is currently being developed for the treatment of type 2 diabetes and obesity [36]. Adiponectin is an anti-diabetic and anti-atherogenic adipokine that binds to adiponectin receptors AdipoR1 and AdipoR2, and exerts anti-diabetic effects via the activation of the AMPK and PPAR-α pathways. Since AdipoRon is orally active, a therapeutic approach using AdipoR agonists is promising for the treatment of diabetes and other obesity-related diseases. Alternatively, it has been reported that DNP-methyl ester safely decreases hypertriglyceridemia and insulin resistance without systemic toxicity in rats fed a high fat diet [37]. DNP-methyl ester is a liver-targeted derivative of DNP, demonstrating the potential feasibility of dissociating the toxicity of DNP from its efficacy by altering its pharmacokinetics. DNP-methyl ester is a promising new drug candidate for the prevention of nonalcoholic fatty acid liver and type 2 diabetes. In contrast to these compounds, the mechanism of action for Ppc-1 is obscure, and the effective dose range that induces significant weight loss is quite narrow; weight loss occurs at an intermediate dose of 0.8 mg/week/kg (group B mice), with less weight loss at higher doses of 4 mg/week/kg and 10 mg/week/kg, and no weight loss at a lower dose (0.16 mg/week/kg). Thus, it will be necessary to evaluate the effects of Ppc-1 in mice fed high-fat diets and other fatty liver models, as well as optimize the form of administration including oral and percutaneous routes.