Figure 4.

Functional effects of anti-PLIN1 autoantibodies on lipolysis and lipase activity. Preadipocytes (3T3-L1) were incubated for 180 min with human serum at 1/10 dilution from four patients with AGL (AGL3, AGL13, AGL33, and AGL39) with anti-PLIN1 autoantibodies, one patient without antibodies (AGL19), and a pool of healthy donors. A: Results of the radiometric assessment of basal and stimulated lipolysis. Data represent mean ± SD for triplicates of two independent experiments for each sample. Statistical significance was assessed with one-way ANOVA assay comparing the mean of each column with the mean from untreated cells. B: Results of lipase activity (nanomoles of glycerol), represented as the percentage over maximum activity from cells treated with healthy donor serum. Data represent mean ± SD for triplicates of two independent experiments for each sample. C: Confocal microscopic analysis of mouse preadipocytes revealed colocalization of PLIN1 and IgG from patient AGL39 on the lipid droplet surface (merged image, in yellow). However, no colocalization was observed when using serum from healthy donors (HD). ABHD5 was localized mainly on lipid droplets under treatment with serum from HD or untreated (basal conditions). Under stimulated conditions or in the presence of serum from AGL39, ABHD5 staining was localized on cytosol. DNA was stained with DAPI (blue); IgG binding was detected using FITC-conjugated rabbit anti-human IgG (green); and PLIN1 or ABHD5 was detected with biotin-labeled rabbit IgG followed by Texas Red–labeled streptavidin (red). Scale bars, 20 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.