Liver mRNA expression of Caspl (a), Pycard (b), and Il1[beta] (c); Western blot analysis of caspase-1 and housekeeping [beta]-actin in liver protein extracts from representative animals (d) and quantification of 20 kDa subunit (active caspase-1) relative to [beta]-actin (e) in NFD- and HFD-fed WT mice and P[2x.sub.7][r.sup.-/-] mice (mean [+ or -] SD; n = 4-6 per group).
CML = [N.sup.[epsilon]] (carboxymethyl)lysine; DAMPs = damage-associated molecular patterns; eATP = extracellular ATP; ER = endoplasmic reticulum; FFAs = free fatty acids; IL-1[beta] = interleukin-1 [beta]; LDLs = low density lipoprotein; MVs = microvesicles; NADPH ox = NADPH oxidase; NF-[kappa]B = nuclear factor-[kappa]B; NLRP3 = nucleotide-binding and oligomerization domain (NOD), leucine-rich repeat and pyrin domain containing 3; PR[2X.sub.7] = purinergic receptor [2X.sub.7]; PYCARD = the PYD- CARD adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain; RAGE = receptor for advanced glycation end products; ROS = reactive oxygen species; TLR = toll-like receptor; TNF-[alpha] = tumor necrosis factor-[alpha].
In the peritonitis mouse model (Table 2), PYCARD, IL-6, and lectin-like oxidized-LDL receptor-1 (LOX-1) were significantly upregulated in epididymal and in subcutaneous adipose tissue at all time points, whereas MCP-1 was induced only in epididymal but not in subcutaneous adipose tissue.
In the present study, we could demonstrate a shift in secretion profile from anti-inflammatory adipokines/cytokines such as adiponectin towards proinflammatory adipokines/cytokines such as visfatin, leptin, IL-6, PYCaRd, and MCP-1.
The following primary antibodies were used: WNV (Envelope protein; Abcam, Cambridge, MA), Iba-1 (Wako Chemicals, Richmond, VA), CD3 and Mac-3 (BD Biosciences, San Jose, CA), Fas (X-20, Santa Cruz Biotechnology, Dallas, TX), caspase 3 (Asp175, clone 5A1E, Cell Signaling, Danvers, MA), and PYCARD ASC-speck (MyBiosource, San Diego, CA).
Transcription of other inflammasome components, the NLR family pyrin domain containing 3 (NLRP3) and PYD and CARD domain containing (PYCARD) proteins, was significantly upregulated in OPN KO animals, on day 5 pi compared to baseline (p values = 0.02 and 0.024, resp.) (Figures 5(b) and 5(d)).
The inflammasome is a multiprotein oligomer consisting of caspase-1, PYCARD
, and NALP that mediate the response to cellular danger signals activating and recruiting inflammatory cells.