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. Author manuscript; available in PMC: 2023 Apr 5.
Published in final edited form as: Cell Metab. 2022 Mar 16;34(4):549–563.e8. doi: 10.1016/j.cmet.2022.02.012

Figure 7. Ectopic introduction of the PTPRD ligand-binding-domain (PTPRD-LBD) into the circulation functions as an “asprosin-trap” to ameliorate appetite, body weight and blood glucose in obese mice.

Figure 7.

(a) Asprosin detected by sandwich ELISA in 5nM recombinant asprosin (rAsprosin) preincubated with 500ng GFP or recombinant PTPRD-ligand binding domain (rPTPRD-LBD) for one hour in phosphate buffer saline (two technical replicates of n = 4 biological replicates per treatment). Asprosin levels plotted relative to signal detected in rAsprosin+GFP.

(b-c) Adenoviral vector (Ad5) mediated overexpression of the IL2-tagged PTPRD ligand-binding-domain (Ptprd-LBD) detected at mRNA level in hepatic tissue (b) and as protein in plasma (c) of 18-week-old DIO mice, 15 days post tail vein injection of Ad5-GFP or Ad5-hPTPRD-LBD (1 x 1011 vp/mouse) viruses.

(d) Plasma asprosin levels of 18-week-old DIO mice, 4 days post tail vein injection of Ad5-GFP or Ad5-hPTPRD-LBD (2 x 1011 vp/mouse) viruses. Plasma asprosin plotted relative to day 0 asprosin levels for each mouse (one technical replicate of n = 11 Ad5-GFP and 10 Ad5-hPTPRD-LBD biological replicates).

(e-h) Food intake, body weight change, baseline glucose and glucose tolerance test (GTT) measured in 18-week-old male DIO mice 12-14 days post tail-vein-injection of Ad5-GFP or Ad5-hPTPRD-LBD (1 x 1011 vp/mouse) viruses (body weight change, food intake: Ad5-GFP: n = 9; Ad5-hPTPRD-LBD: n = 10; baseline glucose: n = 10/treatment; GTT: Ad5-GFP: n = 8; Ad5h-PTPRD-LBD: n = 7).

(i-k) Representative action potential firing traces, depolarization and firing frequency of AgRP neurons after recombinant asprosin (rAsprosin), rAsprosin + recombinant PTPRD-LBD (rPTPRD-LBD), and rAsprosin treatment (three technical replicates of n = 6 biological replicates per treatment).

(l-n) Representative action potential firing traces, depolarization and firing frequency of AgRP neurons after rAsprosin and rAsprosin + GFP treatment, to test the effect of irrelevant protein (GFP) treatment (three technical replicates of n = 5 biological replicates per treatment).

(o,p) A representative western blot and relative quantification of β-actin, p-Stat3 and Stat3 in 10μg cell lysate from HEK293T cells transfected with empty-backbone (pEmpty) or asprosin coding (pAsprosin) mammalian expression plasmid (2 μg) for 48 hours, followed by 8 hours of treatment with rGFP or rPTPRD-LBD (1 μg; three technical replicates of n = 4 biological replicates per treatment).

(q) Percent change in Stat3-response element driven luciferase activity measured in HEK293T cells treated with GFP or rPTPRD-LBD (1 μg) for 12 hours, 72 hours post co-transfection of 2μg 4xM67 pTATA-TK-Luc plasmid with 1 μg pEmpty or pAsprosin (pEmpty+ GFP: n = 11; pAsprosin+ GFP: n = 12; pAsprosin+ rPTPRD-LBD: n = 11).

*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; by two-tailed Student’s t-test and 2-way ANOVA (h). Data are represented as mean ± SEM.See also Figure S10.