Extended Data Fig. 3: Adipsin prevents beta cell death and prevents loss of beta cell transcriptional identity.
(a) Representative images of immunofluorescence (IF) staining for insulin and TUNEL assay in pancreases of db/db mice injected with AAV-Adipsin versus controls. White dashed box indicates region magnified in white panel. IF was repeated at least twice independently with similar results. (b) Quantification of TUNEL + beta cells as determined by IF. (n = 4 mice per group at 1.5 months, n = 5 mice per group at 6 months). Data were analyzed by two-tailed unpaired t-test. (p = 0.03). (c) Representative images of immunofluorescence (IF) staining for insulin and Ki67 in pancreases of the indicated groups of mice. White dashed box indicates region magnified in white panel. IF was repeated at least twice independently with similar results. (d) Quantification of Ki67 + beta cells as determined by IF (At 1.5 months; n = 6 mice per group. At 6 months n = 3 db/db-Adipsin, n = 4 db/db-GFP). (e) Glucagon positive cells were quantitated by IHC in the pancreases of db/db mice transduced with AAV-Adipsin versus controls. Representative images are shown. IHC was repeated at least three times independently with similar results. (f) Quantification of alpha cell mass in B6 WT mice, db/db mice treated with AAV-GFP and db/db mice treated with adipsin AAV at the indicated time points (Pre AAV; n = 4 db/db, n = 4 B6 WT. At 1.5 months post-AAV; n = 6 db/db-Adipsin, n = 5 db/db-GFP, n = 2 B6 WT. At 6 months post-AAV; n = 13 db/db-Adipsin, n = 16 db/db-GFP, n = 4 B6 WT). Data were analyzed by two-tailed unpaired t-test. (At 1.5 months post-AAV db/db-GFP vs db/db-Adipsin p = 0.03). (g) Quantification of the ratio between alpha cell mass and beta cell mass in pancreases from the specified groups at the indicated time points (Pre AAV; n = 4 db/db, n = 4 B6 WT. At 1.5 months post-AAV; n = 6 db/db-Adipsin, n = 5 db/db-GFP, n = 2 B6 WT. At 6 months post-AAV; n = 13 db/db-Adipsin, n = 16 db/db-GFP, n = 4 B6 WT). Data were analyzed by two-tailed unpaired t-test. (At 6 months post-AAV db/db-GFP vs db/db-Adipsin p = 0.029). (h) Fasting serum glucagon levels in db/db mice transduced with AAV-GFP or AAV-Adipsin (n = 8 per group). Scale bars, 100 µm (a, c and e). Data are expressed as mean ± s.e.m. *P < 0.05, ** P < 0.01, *** P < 0.001.
