Fig 2: Gene and protein expression levels in THP1 cells treated for twenty-four hours. mRNA (A) and protein (B) expression levels of UCN1, UCN2, UCN3, CRH, and Spexin were respectively measured by quantitative real-time PCR and Western blots in THP1 cells treated with high glucose (25 mM), palmitate (200 µM) and the combination for 24 h (n = 3 for each condition). Data are normalized to internal GAPDH and presented as fold-change compared with control data. One-way ANOVA with repeated measurements followed by Sidak´s post hoc test were used to determine the significance of the differences in means between the groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 3: UCN3 expression levels in subcutaneous adipose tissue (SAT). (A) Representative confocal immunofluorescence images illustrating UCN3 expression and localization in SAT from normal-weight, overweight people with and without T2D (n = 10 for each group). Staining quantification of SAT was performed as detailed in Materials and Methods and data are presented as arbitrary unit. (B) mRNA expression of Ucn3 in the subcutaneous adipose tissue (SAT) of participant groups. mRNA levels were measured by quantitative real-time PCR using SAT from normal-weight and overweight people with and without T2D and PCR products were run on agarose gel. GAPDH was used as an internal control for normalization and data are presented as fold changes compared with the findings in the normal-weight participant. (C) Representative confocal immunofluorescence images illustrating UCN3 expression and localization in SAT from overweight individuals with and without T2D before and after 3-month physical exercise (n = 6 for each group). Staining quantification of SAT was performed as detailed in Materials and Methods and data are presented as arbitrary unit. The p-value was determined using the Wilcoxon test was used for comparisons between each 2 groups separately and a paired t-test for intragroup comparisons before and after exercise. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Gene expression in THP1 cells treated for four hours. mRNA expression levels of UCN1, UCN2, UCN3, CRH, and spexin were measured by quantitative real-time PCR in THP1 cells treated with high glucose (25 mM), palmitate (200 µM) or the combination for 4 h (n = 3 for each condition). Data are normalized to internal GAPDH and presented as fold-change compared with control data. One-way ANOVA with repeated measurements followed by Sidak´s post hoc test were used to determine the significance of the differences in means between the groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig 5: Expression levels of UCN3 and HSPs during the differentiation of 3T3-L1 preadipocytes and their treatment with a macrophage culture medium (MaCM). mRNA expression levels of UCN3, HSP90, GRP78, CHOP, HSP72, and HSP60 were measured by quantitative real-time PCR in 3T3-L1 preadipocytes (d0) and adipocytes differentiated for 8 days (d8) with and without MaCM. GAPDH was used as an internal control for normalization and data are presented as fold changes in differentiated adipocytes compared with preadipocytes from independent experiments (n = 4). *p < 0.05.