Fig 1: Circulating adiponectin and ceramide levels and characterization of the ADIPOQ mutation.a The level of circulating adiponectin, measured by enzyme-linked immunosorbent assay (ELISA), in noncarriers (black, n = 7) and carriers (red, n = 8) of the ADIPOQ mutation (two-tailed t-test, α = 0.05, *p value ≤ 0.05, error bars SEM). b The intensity values of bands from western blotting for noncarrier (black, n = 2) and carrier (red, n = 2) patients. Carriers of the ADIPOQ mutation lack bands in FPLC fractions associated with high molecular weight (HMW) adiponectin complexes (error bars SEM). Representative western blots are shown and area under the curve was calculated for each peak, HMW, low molecular weight (LMW), and adiponectin trimers (2-way ANOVA with Fisher’s LSD correction, α = 0.05, p = 0.018, error bars are SEM). c The levels of circulating adiponectin, measured by ELISA, in noncarriers (black, n = 4) and carriers (red, n = 10) of the ADIPOQ mutation relative to C16.0 ceramide levels, measured by LC-MS/MS. d Western blot showing wild-type and mutant adiponectin produced in HEK293T cells. The upper blot represents cell lysates and the lower blot shows the two proteins excreted into the cell media. The lanes denoted with a+ were samples taken from cells treated with 20 µg/mL of MG132. Beta actin was used as a loading control. e The FRET efficiency calculated from acceptor bleaching and calculating the increase of intensity of the donor fluorophore. Clover-ADIPOQ and mRuby2-ADIPOQ serve as a positive control for protein-protein binding and FRET. The Clover-ADIPOQ and mutant mRuby2-ADIPOQ cells show no significant FRET efficiency, though very little mutant mRuby2-ADIPOQ is observed in the cells. This is ameliorated by the addition of MG132 (20 µg/mL) blocking mutant mRuby2-ADIPOQ from being degraded. The number of cells counted for quantification is denoted above each bar in the graph. (Asterisks represent significance by a one-sample t test indicating that the chance of FRET occurring is above zero, α = 0.05, *p value ≤ 0.05, **p value ≤ 0.01, ****p value ≤ 0.0001. The large star represents a significant difference between non-treated and MG132 treated Clover-ADIPOQ and mutant mRuby2- ADIPOQ cells by two-tailed t test, α = 0.05, p = 0.04, error bars SEM). The panel also shows an intensity modulated ratiometric image of cells treated with MG132 that produce both Clover-ADIPOQ and the mutant mRuby2-ADIPOQ proteins. Red pseudo color shows pixels in which the donor intensity increases after photobleaching, indicating a FRET signal as the proteins interact.
Fig 2: Relative mRNA (a) and protein (b) expression of adipokines in the SAT and EAT samples. PCR was performed in 55 patients, for Western blots 4 patients in each study group were selected randomly (12 in total). Cyclophillin A was used as a housekeeping gene in PCR, GAPDH was a loading control in Western blots. Quantifications in PCR were made with a Pfaffl method. The Mann–Whitney U test was used to determine differences between fat depots. Each comparison of mRNA expression was statistically significant with p < 0.001; **indicates p < 0.001 in protein expression. EAT epicardial adipose tissue, SAT subcutaneous adipose tissue, APN adiponectin, IL-6 interleukin 6, TNF-α tumor necrosis factor α, GAPDH glyceraldehyde 3-phosphate dehydrogenase
Fig 3: Intranasal administration of Adiponectin improved short-term neurological function at 72 h post-GMH. (A) Righting reflex and (B) Geotaxis reflex tests revealed that the medium dosage (0.1mg/kg) of rh-APN significantly improved neurological function when compared with the vehicle-treated pups at 1, 2, and 3 d post-GMH. Values are expressed as mean ± SD. ANOVA, Dunnett. n = 6-9 for each group. *P < 0.05 compared to sham, #P < 0.05 compared to GMH + Vehicle, @P < 0.05 compared to GMH + rh-APN (0.05mg/kg), &P < 0.05 compared to GMH + rh-APN (0.1mg/kg). (C) Western blot data showed that intranasal administration of rh-APN after GMH could be delivered successfully into brain tissue, and it reduced proinflammatory cytokine levels of IL-1β and IL-6 while increasing expression levels of anti-inflammatory cytokine, IL-10, within the brain. ANOVA, Dunnett. n = 6 for each group. *P < 0.05 compared to sham, #P < 0.05 compared to GMH + Vehicle. (D) Immunofluorescence staining assay showed IL-6 immunoreactivity in the choroid plexus was dramatically increased in pups that were subjected to GMH when compared with sham, and rh-APN treatment significantly reduced the IL-6 immunoreactivity compared to GMH.
Fig 4: rh-APN administration improved long-term memory and motor function at 21 to 28 days after GMH. Water maze test (A–E) showed that rh-APN treatment significantly improved spatial learning and memory performance with reduced swim distance to find the platform (A), less time to escape (B), and more time spent in the probe quadrant (C, D). However, no significant difference in swim velocity was found among the three groups (E). rh-APN treatment notably improved motor function of pups assessed by rotarod (F) and foot fault (G) tests after GMH. (H) Histograms showing the weight changes of pups with or without rh-APN treatment in 4 weeks post-GMH. Values are expressed as mean ± SD. ANOVA, Dunnett. n = 8 for each group. *P < 0.05 compared to sham, #P < 0.05 compared to GMH + Vehicle.
Fig 5: rh-APN treatment promoted M2 microglia polarization at 72 h post GMH. (A) Representative images of immunofluorescence staining and quantification (B, C) depicting co-localization of CD68 (M1 marker, red) or CD206 (M2 marker, red) and Iba-1 (green). Scale bar = 50μm. (D) Representative image of Western blot data showing the expression of CD68, CD206, PPARγ, and CD36. (E-H) Western blot quantification showed that the expression of CD68 was markedly increased and CD206, PPARγ, and CD36 expression were slightly increased compared to sham after GMH. (D–H) rh-APN significantly decreased CD68 expression, while increased the expression of CD206, PPARγ, and CD36. (D–H) However, these effects were reversed by knockdown of AdipoR1 with AdipoR1 siRNA. Values are expressed as mean ± SD. ANOVA, Dunnett. n = 6 for each group. *P < 0.05 compared to sham, #P < 0.05 compared to GMH + Vehicle, @P < 0.05 compared to GMH + rh-APN or GMH + rh-APN + Scramble siRNA.
Supplier Page from Abcam for Anti-Adiponectin antibody [EPR3217]