Metabolic alterations and distribution of rat liver lysosomes in colloidal silica

Abstract

Lysosome-enriched samples from normal fed rat livers have been shown to separate into dense and buoyant bands when layered on self-generating gradients of colloidal silica. The hypothesis that the buoyant peak is comprised largely of autophagic vacuoles that are selectively restrained by sedimenting mitochondria was tested in livers subjected to conditions known to accelerate autophagy or suppress it. In all cases, a sharp peak of beta-N-acetyl-D-glucosaminidase activity was evident when autophagy was present but was undetectable when it was eliminated or virtually eliminated. Moreover, the sensitivity to osmotic shock of buoyant peak particles, but not those of the dense band, was increased after autophagic stimulation. The total amount of enzyme that moved to buoyant fractions with the induction of autophagy generally correlated with the degree of autophagic acceleration. However, with maximal autophagy, the buoyant peak was broad, and only 70% was capable of being dislodged and returned to the dense peak by dispersing samples in the gradient medium and/or reducing the sample size. On the other hand, at roughly half-maximal stimulation and lower, the buoyant distribution was sharply defined and completely reversible. These findings support the above hypothesis and suggest that the buoyant peak could be used as a measure of autophagy in the physiological range of regulation.

Identification of Autolysosomes Directly Associated with Proteolysis on the Density Gradients in Isolated Rat Hepatocytes

“…As shown in Fig. 4A, when the M + L fraction was dispersed through the gradient medium before centrifugation, a small portion of vacuolar particles in the buoyant peak moved down to the dense region, which was the same response as that in the liver (10,11,20). The behaviour of soluble, non-particulate materials in these two modes was monitored by u C-sucrose (Fig.…”

“…The profiles showed a remarkable contrast. The liver tissue exhibited a large peak in the dense region just as in Ludox gradients (10,11), whereas hepatocytes exhibited a smaller peak in the dense region and a much larger peak in the buoyant region. Therefore, the difference in the profiles is due not to the gradient medium, but to autophagic/lysosomal particles in the preparation.…”

“…Rome et al improved separation by employing a mixture of Ludox, a colloidal silica, and PVP (25). Surmacz et al were the first to apply this method successfully to autophagy-related particles (10,11). Since then, the colloidal silica has been found most effective for separating two lysosomal populations, AV and secondary lysosomes, compared with other gradient media, e.g., metrizamide or Nycodenz.…”

“…A mixture of 60% Percoll and 0.75% PVP was finally chosen, in which the profile consisted of a major buoyant peak (d= 1.090) and a minor dense peak (d= 1.131), quite similar to the profile obtained previously (19). However, this /9-hexosaminidase profile obtained from isolated hepatocytes was quite different from that in liver perfusion study (10,11,20). In order to test if this was the result of a difference in gradient media or lysosomal population in these two preparations, they were directly compared under the same gradient conditions (Fig.…”

“…For the present study, which aims to separate subpopulations among autophagy-related vacuoles rather than to purify them from other organelles such as mitochondria, a colloidal silica was chosen. Previous studies (10,11) demonstrated that autophagic vacuoles (AV) induced in the perfused liver could be effectively separated from DB using Ludox, a colloidal silica, mixed with polyvinylpyrrolidone (PVP) , and this has been regarded as the best separation technique of AVd and DB, intermediate and late compartments of autophagy, respectively. We applied this method to freshly isolated rat hepatocytes using Percoll, a more readily available colloidal silica than Ludox.…”