Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation

Figures

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  Figure 1. Biophysical characterization of α-Clu and β-Clu. (a) Far-UV CD spectra of α-Clu (- - - -) and β-Clu (….). [θ]MRE represents the mean residue ellipticity. (b) Intrinsic tryptophan fluorescence spectra of 0.2 mg/ml of α-Clu (- - - -) and β-Clu (….) in PBS. Excitation wavelength was set at 295 nm and the excitation and emission band passes were set at 2.5 nm. (c) Fluorescence spectra of the hydrophobic fluorescent probe, bis-ANS, alone (-----) or in the presence of α-Clu (- - - -) or β-Clu (….). The excitation wavelength was set at 390 nm and excitation and emission band passes were set at 2.5 nm. (d) DLS population distribution for α-Clu (i) and β-Clu (ii). The distribution obtained is an average of 3 sets of 20 acquisitions each. The abscissa indicates the hydrodynamic radius of the molecule, ordinate is relative abundance of the molecules and the area under the curve indicates the polydispersity. (e) Distribution of sedimentation coefficients of α-Clu. The molecular masses of the species determined from the sedimentation velocity data by solving Lamm’s equation are also indicated.

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  Figure 2. Chaperone activity of α-Clu (- - -) and β-Clu (…) against (a) DTT induced aggregation of insulin (-.-.-) and (b) Heat induced aggregation of yeast ADH (-.-.-). Control traces with α-Clu alone () and β-Clu alone () are shown in both panels.

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  Figure 3. Sub cellular localization of α-Clu and β-Clu. C2C12 cells were incubated with FITC- α-Clu or FITC-β-Clu for 24 hours, followed by incubation with Lysotracker Red for 1 hour. Cells were visualized on Leica SP8 Confocal platform. Excitation of He-Ne diode of 405 nm for DAPI and Argon laser lines of 488 nm and 560 nm for FITC and Lysotracker respectively were used. (a) α-Clu; (b) β-Clu. Represented images are single mid-sections of observed fields. Scale bar represents 10 μm.

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  Figure 4. Lipid accumulation in cell lines. Control cells (i), α-Clu-treated cells (ii) and β-Clu-treated cells (iii) stained with Nile Red (Panel a: C2C12; Panel b: F111) and Oil Red O (Panel c: C2C12; Panel d: F111). Solid black arrows in c and d represent morphological changes observed in the cells upon incubation with β-Clu. Scale bar in panels a and b represent 50 μm. Scale bars in panels c and d represent 40 μm.

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  Figure 5. Lipid accumulation in cells upon β-Clu administration is a general phenomenon. Oil red O staining of control cells (i) and cells treated with α-Clu (ii) and β-Clu (iii). Morphological changes are represented by arrows. Hypertrophy in 3T3-L1 preadipocyte cells and HepG2 and F111 cells are represented by white arrows. Changes in cellular shape in the cells treated with β-Clu are denoted by black arrows. Scale bar represents 40 μm.

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  Figure 6. Morphological changes and time-dependant accumulation of lipids in cells treated with clusterin chains. (a) Morphology of cells treated with (i) β-Clu alone and (ii) both α-Clu and β-Clu at equimolar ratios. (b) Time-dependant increase in lipid accumulation in β-Clu-treated cells over control and α-Clu treated cells and rescue of phenomena upon addition of equimolar ratios of α-Clu and β-Clu, quantified by FACS (*p = 0.008, two-tailed T-test).

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  Figure 7. Weight change and feed intake of rabbits injected with α-Clu or β-Clu. (a) Average percentage increase in weight of rabbits injected with β-Clu over controls and α-Clu-injected rabbits (n = 8). Red arrows indicate times of booster injections. (b) Average daily feed intake in controls (n = 8) and rabbits injected with α-Clu (n = 8) or β-Clu (n = 8). Red horizontal bar represents mean value. Red + represents median.

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  Figure 8. Changes in organs and tissues of rabbits injected with β-Clu. (a) Fatty changes in liver of β-Clu-injected rabbit evident as focal patchy yellowish discoloration, depicted by red arrows. (b) Fatty changes in kidney of β-Clu-injected rabbit depicted by pale colour of the kidney compared to control. Histological examination of (i) control rabbits, (ii) α-Clu-injected rabbits and (iii) β-Clu-injected rabbits; (c) Adipose tissue, (d) Kidney, (e) Liver. Double sided arrows indicate relative hypertrophy in β-Clu-injected rabbits with respect to control and α-Clu-injected rabbits. Black arrows indicate extensive steatosis in liver tissues of β-Clu injected rabbits. Scale bar represents 40 μm.