Figure 1.
Bacterial PGN stimulates lipolysis via NOD1.
Gonadal WAT explants from WT mice were incubated with various doses of FK565 and glycerol concentration in the media determined at 0, 24, 48 and 72(A). Glycerol release rate over 72 h was calculated in response to various doses of FK565 (NOD1 ligand) or MDP (NOD2 ligand) in WAT explants from WT mice (B). Gonadal WAT explants from WT or NOD1−/− mice were incubated without (Control) or with FK565 (10 µg/mL) and glycerol concentration in the media determined at 0, 24, 48 and 72 h (C). Glycerol release rate over 72 h was calculated in explants from WT or NOD1−/− mice, which were incubated with or without FK565 (D). Gonadal WAT explants from WT or NOD1−/− mice were incubated with FK565 (10 µg/mL) and NEFA concentration in the media was determined at 72 h (E). n = 6–12 explants per condition, values are mean ± SEM. *Significantly different from the WT control condition or 0 µg/mL (i.e. no FK565) at a given time point.
Figure 2.
NOD1-activating PGN causes adipocyte-autonomous lipolysis.
Differentiated 3T3-L1 adipocytes were incubated with FK565 (10 µg/mL) and glycerol (A) and NEFA (B) concentration in the media determined at 0, 24, 48 h. Glycerol release rate over 48 h was calculated in 3T3-L1 adipocytes, which were incubated with vehicle (control), c12-iEDAP (10 µg/mL), FK565 (10 µg/mL) and various doses of MDP (C). Glycerol release rate over 48 h in 3T3-L1 adipocytes without and with FK565 (10 µg/mL) in the absence or presence of the NOD1 inhibitor ML130 (1-[(4-Methylphenyl)sulfonyl]-1H-benzimidazol-2-amine) at various concentrations (D). Glycerol release rate in 3T3-L1 adipocytes that were incubated with or without FK565 (10 µg/mL) and various concentrations of the HSL inhibitor CAY10499 (E). n = 5–16 experiments per condition, values are mean + SEM. *Significantly different from control or control at a given time point. #Significantly different from c12-iEDAP.
Figure 3.
NOD1-activating PGN induces ERK and PKA substrate signals in adipocytes.
Differentiated 3T3-L1 adipocytes were incubated with or without FK565 (10 µg/mL) for 6 and 24 h and phosphorylated ERK1/2Thr202/Tyr204 (pERK) relative to total ERK (A) and phosphorylated p38 relative to total p38 (B) protein levels were determined in cell lysates. 3T3-L1 adipocytes were incubated with or without FK565 (10 µg/mL, 48 h) or with isoproterenol (ISO, 1 µM, 30 min) and serine/threonine phosphorylated PKA substrates (consensus sequence: RRXS/T) were probed in cell lysates (C). Phospho-PKA substrates immunoblot is indicative of four independent replicates and protein loading was measured with β-Actin immunoblotting (C). n = 4–6 experiments per condition, values are mean + SEM. *Significantly different from control.
Figure 4.
NOD1-mediated lipolysis occurs via both ERK and PKA.
Glycerol release rate over 48-L1 adipocytes, which were incubated without or with FK565 (10 µg/mL) and various concentrations of the PKA inhibitor, PKI (14–22) (A). Glycerol release rate over 48 h was calculated in 3T3-L1 adipocytes, which were incubated without or with FK565 (10 µg/mL) and various combinations of H89 (20 µM, PKA inhibitor), and U0126 (10 µM, ERK1/2 inhibitor) or SP600125 (20 µM, JNK inhibitor) (B). 3T3-L1 adipocytes were incubated with or without FK565 (10 µg/mL) for 6 h and phosphorylated ERK1/2Thr202/Tyr204 (pERK) relative to total ERK was determined in the presence or absence of the PKA inhibitors PKI and H89 (C). n = 6–16 experiments per condition, values are mean + SEM. *Significantly different from control without FK565. #Significantly different from control with FK565. φSignificantly different from H89 or U0126 alone with FK565.
Figure 5.
NF-κB is involved in NOD1-mediated lipolysis.
NF-κB activity in 3T3-L1 adipocytes after various exposure times of FK565 (10 µg/mL) (A). NF-κB activity in 3T3-L1 adipocytes without and with 6h exposure to FK565 (10 µg/mL) in the presence and absence of PKI (20 µM) and H89 (20 µM) (B). NF-κB activity in 3T3-L1 adipocytes without and with 6h exposure to FK565 (10 µg/mL) with various doses of U0126 (C). n = 4-6 experiments per condition for NF-κB activity. Glycerol release rate over 48 h in 3T3-L1 adipocytes incubated without or with FK565 (10 µg/mL) with various concentrations of NF-κB inhibitors, PDTC (D) and CAY10470 (E). Glycerol release rate over 48 h in 3T3-L1 adipocytes incubated without or with FK565 (10 µg/mL) with various combinations of H89 (20 µM, PKA inhibitor), and U0126 (20 µM, ERK1/2 inhibitor) or CAY10470 (NF-κB inhibitor, 5 nM) (F). n = 12–24 experiments per condition of lipolysis. Values are mean + SEM. *Significantly different from control without FK565. #Significantly different from control with FK565. φSignificantly different from CAY10470 or U0126 or H89 alone with FK565. εSignificantly different from CAY10470 plus U0126 or CAY10470 plus H89 with FK565.
Figure 6.
Working model of bacterial PGN-mediated lipolysis in adipocytes.
The bacterial peptidoglycan motifs that activate NOD1cause cell autonomous lipolysis in adipocytes. NOD1-activating PGN engages ERK, PKA and NF-κB signals leading to lipolysis. NOD1-mediated signals converge on HSL to stimulate adipocyte lipolysis. Our evidence suggests that separate engagement of ERK and PKA pathways are needed to fully activate NOD1-mediated lipolysis in adipocytes.