Fig 2: Rap1 suppresses lysosome abundance and peripheral distribution upon limitation in amino acids.a–j Effect of Rap1 activity on lysosome distribution and abundance. In a, g representative z-stack projections of endogenous lysosome distribution are shown for U2OS cells expressing wild-type (WT) or G12V Rap1A+B cDNA (a), or after 4 h of amino acid depletion (-AA 4 h) in combination with Rap1A+B depletion (g). The fraction of endosomes in the peripheral and perinuclear region was quantified in an automated fashion (b, c, d, h). Rab5 immunostainings are shown in Supplementary Fig. 3h. The percentage change in lysosomal abundance was quantified using 3D-reconstructions analysis of lysosomes (e, f, i, j). k, l Representative Z-stack projections showing increased lysosome staining in nutrient fed Rap1A+B-depleted HEK293A cells (k). Bar graph shows relative change in average LAMP2 intensity across three individual experiments for 57–72 cells. The ratio between perinuclear and peripheral lysosomes in nutrient fed Rap1A+B-depleted cells is quantified in l. The change in lysosome abundance is shown in Supplementary Fig. 4a, b. Cell boundaries are depicted in yellow and cell nuclei (DAPI) is shown in blue. Scalebars: a, g, k = 10 µm, e, i = 20 µm. The microscopic fields imaged were randomly selected. Experiments were repeated at least three times. In b, d, h, l n denotes the number of individual cells analyzed across three independent experiments and data are presented as mean values ± s.d. In f, j, k n denotes the number of individual experiments and data are presented as mean values ± s.e.m. The number of cells analyzed to quantify lysosome abundance is shown in Supplementary Fig. 14. n.s. = not significant (P > 0.05); Student’s t-test; two-sided, unpaired (d, f, j, k), one-way ANOVA with Tukey’s post hoc test (b, h, l). In h p = 0.8148 and 0.8994 for siRNA Ctrl vs Rap1A+B #1 and #2 peripheral lysosomes, and 0.5731 and 0.585 for siRNA Ctrl vs Rap1A+B #1 and #2 for perinuclear lysosomes, respectively. In b p = 0.9927 and 0.9819 for Ctrl vs Rap1 WT, peripheral and perinuclear fractions, respectively. ***P < 0.001, **P < 0.01 one-way ANOVA with Tukey’s post hoc test (h). See Source Data File for statistics source data.

Fig 3: Actin remodeling contributes to gluconeogenesis and glucose production(A) Primary mouse hepatocytes (HCs) that were treated with scrambled control (scr), si-Rap1a, or cytochalasin D were stained with 488-phalloidin (green) to visualize F-actin. White arrows indicate fragmented F-actin filaments that form cytoskeletal clumps. Scale bar, 10 µm.(B) Hepatocytes were treated with vehicle, 0.1 µM, or 1 µM cytochalasin D (cyto. D) for 8 h, and forskolin and dexamethasone (F + D)-stimulated G6pc and Pck1 mRNA levels were measured (n = 4 biological replicates, mean ± SEM, *p < 0.05).(C) Same as in (B) except that glucose production was measured in cells treated with vehicle or 0.1 µM cytochalasin D (cyto. D) (n = 3 biological replicates, mean ± SEM, *p < 0.05).(D) Hepatocytes were treated with 0.1 µM jasplakinolide (jasp.) for 6 h, and F + D-stimulated G6pc and Pck1 mRNA levels were measured (n = 3 biological replicates, mean ± SEM, *p < 0.05).(E) Same as in (D), except that glucose production was measured (n = 4 biological replicates, mean ± SEM, *p < 0.05).(F) WT and Foxo1,3,4-/- hepatocytes were treated with vehicle or 1 µM cytochalasin D for 8 h, and F + D-stimulated G6pc and Igfbp1 mRNA levels were measured (n = 4 biological replicates, mean ± SEM, *p < 0.05, n.s., non-significant).(G) Nuclear and whole-cell FoxO1 levels along with loading controls (nucleophosmin, Np, and ß-actin, respectively) were assayed in hepatocytes that were treated with vehicle or 1 µM cytochalasin D for 8 h (n = 3 biological replicates).(H) Same as in (G), except that cells were stimulated with insulin, and p-Akt, total Akt, and ß-actin levels were assayed (n = 3 biological replicates).

Fig 4: Activation of Rap1a is suppressed in obese mice liver, and restoring Rap1a activity lowers blood glucose and improves glucose intolerance in obese mice(A) Livers from db/db and control (db/+) mice were assayed for GTP-bound (active) Rap1a and total Rap1a. Densitometric quantification of the immunoblot data is shown in the bar graph (n = 5 mice/group, mean ± SEM, *p < 0.05).(B) Livers from DIO mice and their low-fat-fed controls (lean) were assayed for GTP-bound (active) Rap1a, total Rap1a, and ß-actin. Densitometric quantification of the immunoblot data is shown in the bar graph (n = 6 mice/group, mean ± SEM, *p < 0.05).(C–G) Body weight before and after AAV injection (C), overnight fasting blood glucose (D), glucose tolerance test (E), liver G6pc and Pck1 mRNA (F), and overnight fasting plasma insulin (G) from db/db mice that were injected with adeno-associated viruses (AAV) containing either hepatocyte-specific CA-Rap1a (constitutively active Rap1a) or the control vector (Gfp) (n = 4–7 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant).(H–L) Body weight before and after adenovirus injection (H), 5-h fasting blood glucose (I), glucose tolerance test (J), liver G6pc mRNA (K), and 5-h fasting plasma insulin (L) levels from DIO mice that were injected with adenovirus vectors containing CA-Rap1a or control LacZ (ß-galactosidase) (n = 6 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant).

Fig 5: Statins inhibit Rap1a in isolated hepatocytes and the human liver(A) Primary mouse hepatocytes (HCs) were treated with vehicle or 10 µM simvastatin (simva) for 20 h. Membrane proteins were assayed for Rap1a and Na, K-ATPase (loading control) (n = 2 biological replicates).(B) Same as in (A), except that 5 µM rosuvastatin (rosuva) was used, and membrane fractions and whole-cell lysates were assayed for Rap1a and loading controls (Na, K-ATPase and µ-actin, respectively) (n = 3 biological replicates).(C) Hepatocytes treated as in (B) were assayed for GTP-bound (active) Rap1a and total Rap1a (n = 2 biological replicates).(D) Liver tissue samples from patients on statin therapy and their sex-, age-, and disease-matched controls without statin therapy were assayed for GTP-bound (active) Rap1a and total Rap1a (n = 4 human liver samples/group).(E) Same as in (D), except that livers from another set of patients were assayed for GTP-bound (active) Rap1a and total Rap1a (n = 2 human liver samples/group).