FIGURE 4.

Metformin interferes with autophagy in the intestine during starvation. A–D, after 9 days of starvation, both wild-type worms and nhx-5 mutants showed GFP-LGG-1 puncta in the intestine (A and C, 0 mm metformin). The number of puncta was greatly reduced in wild type by metformin treatment but not as much in nhx-5 mutants (B and D, 100 mm metformin). Yellow arrowheads show that the overall intensity of GFP-LGG-1 in the terminal bulb is not significantly reduced in all the animals. Scale bar = 20 μm. The left panel shows DIC images, and the right panel shows the corresponding GFP images. E, enlarged images of DIC, GFP, and both (from left to right) of the red box in panel A. These images show an example of how individual GFP-LGG-1 puncta looked and how they were counted. The graphic next to the red box in the merged image was drawn to help visualization of individual puncta. A vesicle not labeled by GFP-LGG-1 is marked as a black circle. The yellow arrowhead indicates a birefringent gut granule (no GFP and not an autophagosome), known to be stained with lysosomal dyes (48). GFP-LGG-1 puncta were counted under a GFP filter (GFP, right panel, 63×). DIC (left panel, 63×) is used to show the animals as well as to validate the location of the individual puncta. F, GFP-LGG-1-labeled puncta from starved worms were determined at the given time. Metformin-treated wild type (wt met) showed a significantly reduced number of puncta when compared with the nhx-5 mutants in the presence or absence of metformin (p < 0.05, unpaired two-sample t test). In addition, metformin-treated nhx-5 showed a significantly reduced number of puncta when compared with cohorts in the absence of metformin (p < 0.01). However, wild type and nhx-5 controls are not different in starvation-induced GFP-LGG-1-labeled puncta. To avoid a sampling error, the days were cut off when fewer than 30% of animals were alive because the dead animals did not show autophagy puncta. The numbers are the mean ± S.E.