Rab7 is localized on ZG membranes in wild-type mouse pancreatic acinar cells.
First, we examined the intracellular localization of Rab7 in mouse pancreatic acinar cells. As shown in Fig. 1a, immunofluorescence microscopy revealed that Rab7 colocalized with amylase in pancreatic acinar cells of wild-type mice (Fig. 1a, left column). In contrast, no discernible Rab7 signals were observed in Rab7Δpan pancreatic acinar cells (Fig. 1a, right column). These data indicate that Rab7 is localized to ZGs in mouse pancreatic acinar cells. Of note, the immunofluorescence signal of Rab7 does not completely overlap with that of amylase, implying that Rab7 is also localized to other intracellular organelles, such as late endosomes and lysosomes.
In addition, Western blotting of Rab7 using highly purified ZG membranes confirmed Rab7 localization on ZG membranes (Fig. 1b).
Rab7 is not involved in basal or CCK-stimulated exocytosis.
Based on Rab7 localization to ZGs, we hypothesized that Rab7 might be involved in the regulation of exocytosis. To test this hypothesis, we compared stimulated amylase secretion using isolated pancreatic acini between wild-type and Rab7Δpan mice. As shown in Fig. 2, no apparent difference was observed in either the basal or stimulated amylase secretion from isolated pancreatic acini between wild-type and Rab7Δpan mice. These data indicated that Rab7 localized on ZGs does not function in the regulated exocytosis of pancreatic acinar cells.
The diameter of ZGs is significantly smaller and the number of ZGs is increased in Rab7 Δpan than in wild-type mice.
To further elucidate the function of Rab7 localized on ZGs, we examined the effect of Rab7 knockout on the size of ZGs using electron microscopy. As shown in Fig. 3, the sizes of ZGs in Rab7Δpan are significantly smaller than those in wild-type mice under fed conditions (Fig. 3a-d). These data indicate that Rab7 on ZGs plays a positive role in preserving the size of the ZGs.
Next, we investigated the effects of Rab7 disruption on the number of ZGs. As shown in Fig. 4a, under fed conditions, the number of ZGs was markedly increased (approximately 1.6-fold) in the pancreatic acinar cells of Rab7Δpan compared with that in wild-type mice. Given that the diameter of ZGs was reduced concomitantly with an increase in their number in Rab7Δpan, these data strongly suggest that Rab7 promotes ZG maturation.
Rab7 is not essential for the ZG degradation by starvation-induced autophagy.
Although it has been previously reported that ZGs are degraded by starvation-induced autophagy 14, the molecular mechanism is still uncertain. Rab7 is also known to function in autophagy4. Therefore, we examined whether or not Rab7 in ZGs participates in ZG degradation by starvation-induced autophagy. To this end, we investigated the effect of starvation on the number of ZGs using electron microscopy. As shown in Fig. 4a, electron microscopic observation demonstrated that 24 h of starvation decreased the number of ZGs compared to that under fed conditions in both Rab7Δpan and wild-type mice. These data indicated that Rab7 is dispensable for the degradation of ZGs by starvation-induced autophagy.
To confirm the dispensation of Rab7 in starvation-induced autophagy, we compared the amounts of amylase, a representative and major digestive enzyme packed in ZGs, in the pancreas before and after starvation between Rab7Δpan and wild-type mice using immunohistochemistry and Western blotting. As shown in Fig. 4b, amylase immunoreactivity in the pancreas before starvation was comparable between Rab7Δpan and wild-type mice, implying that disruption of Rab7 does not affect the amount of digestive enzymes synthesized in pancreatic acinar cells. After 24 h of starvation, immunoreactivity to amylase in the pancreas decreased in both Rab7Δpan and wild-type mice compared to that in fed conditions. Western blotting confirmed these results, and starvation decreased the amount of amylase significantly in both Rab7Δpan and wild-type mice (Fig. 4c). The amounts of amylase before and after starvation were comparable between the Rab7Δpan and wild-type mice. These data confirm that Rab7 is not essential for the degradation of ZGs by starvation-induced autophagy.
Cathepsin B is activated by the degradation of LAMP1 in Rab7 Δpan .
In our previous study, lysosomal functions were affected in Rab7Δpan, and the autophagy flux was impaired between the autophagosome and autolysosome processes during starvation8. Lysosomal enzymes play a key role in the degradation of ZGs by starvation-induced autophagy14. However, starvation-induced degradation of ZGs was not altered in Rab7Δpan. We therefore compared the lysosomal function under fed and starved conditions.
Lysosomal functions, including the expression of cathepsin B, a representative lysosomal protease, and that of LAMP-1, which is essential for lysosomal functions15 at fed conditions, were evaluated in our previous study8. To determine the effect of Rab7 disruption under starved conditions on these lysosomal functions and to compare the changes between both conditions, we evaluated the lysosomal functions under fed and starved conditions simultaneously. As shown in Fig. 5a, the cathepsin B expression in Rab7Δpan was already increased under physiological feeding conditions and was comparable to that under starved conditions. Likewise, in Western blotting (Fig. 5b), the expression of the double-chain form (a mature active form) of Cathepsin B was much elevated in Rab7Δpan pancreas under both fed and starved conditions. Immunohistochemistry (Fig. 5a) showed that the immunoreactivity of LAMP-1 was fairly increased in Rab7Δpan compared to the wild type in both fed and starved conditions. The expression was stronger around the perinuclear region, suggesting that lysosomal localization was not altered in Rab7Δpan. Western blotting showed that the band of LAMP-1 shifted to a lower molecular position with laddering in Rab7Δpan under both fed and starved conditions, suggesting degradation of LAMP-1 in Rab7Δpan. The extent of degradation appeared to be stronger under the starved condition than under the fed condition.
Cathepsin B converts trypsinogen to trypsin, whereas cathepsin L degrades both trypsinogen and trypsin. To confirm the overexpression of cathepsin in Rab7Δpan, we evaluated the activity of cathepsins B and L under basal feeding conditions using a fluorometric assay kit. As shown in Fig. 5c, the activities of cathepsin B and L were significantly elevated in Rab7Δpan compared to wild-type mice.