Table 1.
Basal clinical and biochemical characteristics of subjects.
Figure 1.
Proteomic evaluation predicts adiponectin isoforms as being differentially expressed between VDD and NVD.
A 2D-electrophoretic analysis was performed in duplicate for the 42 subjects using IPG3-10, with proteins detected by Sypro Ruby staining. Spot/s predicted to be adiponectin are indicated by the PDQuest identification number (SSP3050). Supportive evidence for the prediction was given by performing a WIB of 2D-electrohoretic gels using anti-adiponectin antibody. Representative gels are shown.
Table 2.
Top ten most significantly modulated plasma proteins between VDD and NVD obese pediatric subjects.
Figure 2.
WIB of proteomic analyses and plasma samples confirms that total adiponectin is decreased in VD deficient pediatric obese subjects (VDD) when compared to pediatric obese with normal VD levels (NVD).
A. A WIB of 2D-electrophoretic analyses was performed in VDD (n = 10) and NVD (n = 10) subjects using anti-adiponectin antibody. B. A WIB analysis under reduced conditions of total adiponectin in the plasma of representative VDD (<15 ng/ml) and NVD (>30 ng/ml) subjects.
Figure 3.
The multimeric forms of adiponectin are reduced, in particular the HMW form, in VD deficient pediatric obese subjects.
A WIB analysis under non-reduced conditions and a quantitative densitometric analysis of the multimeric forms of adiponectin (HMW, MMW, LMW) in the plasma of representative VDD (<15 ng/ml; n = 18) and NVD (>30 ng/ml; n = 24) subjects. Densitometric results were normalized to plasma protein concentrations.
Figure 4.
Total and the HMW and MMW multimeric forms of adiponectin increase in VD deficient pediatric obese subjects following cholecalciferol supplementation for 12mo.
A. A WIB analysis under reduced conditions and a quantitative densitometric analysis of total adiponectin in the plasma of representative VDD (<15 ng/ml) and NVD (>30 ng/ml) subjects. B. A WIB analysis under non-reduced conditions and a quantitative densitometric analysis of the multimeric forms of adiponectin in the plasma of VDD (<15 ng/ml; n = 18) and NVD (>30 ng/ml; n = 24) subjects.
Figure 5.
Total adiponectin secretion increases in 3T3-L1 adipocytes treated with 1α,25-(OH)2D3.
3T3-L1 adipocytes, generated using a standard differentiation protocol, at day 10 were treated with increasing concentrations of 1α,25-(OH)2D3 (10−9 to 10−7 M) in SFM or SFM with vehicle for 48 h. The CM at 7, 24 and 48 h from the same treatment was analyzed by WIB under reduced condition and analyzed densitometrically for the synthesis of adiponectin. Results were normalized to α-tubulin and are presented as fold-increase with respect to the 7 h SFM sample (n = 4).
Figure 6.
The secretion of the adiponectin multimeric forms, in particular the LMW form, in parallel with the ER-chaperon DsbA-L, increases in 3T3-L1 adipocytes treated with 1α,25-(OH)2D3.
3T3-L1 adipocytes, generated using a standard differentiation protocol, at day 10 were treated with increasing concentrations of 1α,25-(OH)2D3 (10-9 to 10-7M) in SFM or SFM with vehicle for 48 h. The CM at 7, 24 and 48 h from the same treatment and CL at 48 h, was analyzed by WIB under non-reduced or reduced conditions for the synthesis of adiponectin multimeric forms or DsbA-L with α-tubulin, respectively.