Carnosic acid (CA) prevents lipid accumulation in hepatocytes through the EGFR/MAPK pathway.

BACKGROUND: Carnosic acid (CA), found in rosemary, has been reported to have antioxidant and anti-adipogenic properties. We recently demonstrated that CA protects against steatosis in ob/ob mice. In the present report, we investigated the molecular mechanism by which CA inhibits lipids accumulation both in vivo and in vitro. METHODS: In the in vivo study, ob/ob mice were fed a standard chow diet with or without CA for 5 weeks, then their hepatocyte lipid accumulation was determined. The serum concentrations of cytokines, the levels of lipid regulatory mediators, and the hepatic metabolic and signaling molecules were also evaluated. In the in vitro study, HepG2 cells were used to further clarify the effects of CA on cellular lipid accumulation and to confirm the signaling pathways involved in these effects. RESULTS: CA significantly reduced hepatocyte lipid accumulation. This effect was associated with repressed levels of hepatic PPARγ, reduced expression of inflammatory cytokines such as IL-1ß, IL-12, IL-17, IFN-γ, MCP-1, and MIP-1ß, and increased ATP, acetyl CoA, NAD(P)(+), and NAD(P)H. Other signaling molecules, such as EGFR, MAPK, AMPK, and ACC, which regulate lipid metabolism, were activated in mice fed the CA diet. CA inhibited palmitate-induced cellular lipid accumulation and stimulated the phosphorylation of both EGFR and MAPK. Pretreatment with either the EGFR inhibitor AG1478 or the MEK-specific inhibitor U0126 abolished the effects of CA on cellular lipid accumulation and decreased both the protein expression and activity of PPARγ. CONCLUSIONS: EGFR/MAPK signaling plays an important role in the inhibitory effect of CA on hepatocyte lipid accumulation.

Dual neuroprotective pathways of a pro-electrophilic compound via HSF-1-activated heat-shock proteins and Nrf2-activated phase 2 antioxidant response enzymes.

Activation of the Keap1/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and consequent induction of phase 2 antioxidant enzymes is known to afford neuroprotection. Here, we present a series of novel electrophilic compounds that protect neurons via this pathway. Natural products, such as carnosic acid (CA), are present in high amounts in the herbs rosemary and sage as ortho-dihydroquinones, and have attracted particular attention because they are converted by oxidative stress to their active form (ortho-quinone species) that stimulate the Keap1/Nrf2 transcriptional pathway. Once activated, this pathway leads to the production of a series of antioxidant phase 2 enzymes. Thus, such dihydroquinones function as redox-activated 'pro-electrophiles'. Here, we explored the concept that related para-dihydroquinones represent even more effective bioactive pro-electrophiles for the induction of phase 2 enzymes without producing toxic side effects. We synthesized several novel para-hydroquinone-type pro-electrophilic compounds (designated D1 and D2) to analyze their protective mechanism. DNA microarray, PCR, and western blot analyses showed that compound D1 induced expression of heat-shock proteins (HSPs), including HSP70, HSP27, and DnaJ, in addition to phase 2 enzymes such as hemeoxygenase-1 (HO-1), NADP(H) quinine-oxidoreductase1, and the Na(+)-independent cystine/glutamate exchanger (xCT). Treatment with D1 resulted in activation of Nrf2 and heat-shock transcription factor-1 (HSF-1) transcriptional elements, thus inducing phase 2 enzymes and HSPs, respectively. In this manner, D1 protected neuronal cells from both oxidative and endoplasmic reticulum (ER)-related stress. Additionally, D1 suppressed induction of 78 kDa glucose-regulated protein (GRP78), an ER chaperone protein, and inhibited hyperoxidation of peroxiredoxin 2 (PRX2), a molecule that is in its reduced state can protect from oxidative stress. These results suggest that D1 is a novel pro-electrophilic compound that activates both the Nrf2 and HSF-1 pathways, and may thus offer protection from oxidative and ER stress.

Activated glutathione metabolism participates in protective effects of carnosic acid against oxidative stress in neuronal HT22 cells.

In our previous studies, we have reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protects cortical neurons by inducing phase 2 enzymes, the induction of which was initiated by activation of the Keap1/Nrf2 pathway , , . In the present study we address the nature of the effector of these neuroprotective effects downstream of the phase 2 enzyme induction. From our results we conclude that activated glutathione (GSH) metabolism may participate in these protective effects. First, we performed cDNA microarray analysis in order to identify the gene(s) responsible for the actions and found that various enzymes involved in the metabolism of GSH (glutathione S-transferase, alpha 4; glutathione S-transferase, alpha 2; and formylglutathione hydrolase) constituted 3 of the top 5 CA-induced genes. The other 2 genes encoded phase 2 enzymes [NAD(P)H-quinone oxidoreductase1and aldehyde dehydrogenase family 3, subfamily A1]. Next, we compared the physiologically-active compounds originating from rosemary (CA, CS, luteolin, genkwanin, rosmarinic acid, caffeic acid, and verbenone) by 3 criteria (enhancement of total glutathione levels, transcriptional activation, neuroprotective effects). By all of these criteria, CA and CS were the most active. In contrast, the other compounds were only weakly active or totally inactive. These results suggest that pro-electrophilic compounds such as CA and CS may protect cortical neurons by causing the following sequential events: S-alkylation --> activation of the Keap1/Nrf2 pathway --> transcriptional activation --> induction of phase 2 enzymes --> activation of GSH metabolism --> neuroprotection.

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism.

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.