(A) Fed and fasting blood glucose levels (left) and glucose tolerance (right) in LKB1^lox/lox and PLKB mice (n = 11 mice per genotype). Mice were injected with tamoxifen at 4 weeks of age, and assays were performed 1 week later.
(B) Insulin sensitivity in LKB1^lox/lox and PLKB mice (n = 9 mice per genotype). Same mice were used as in (A).
(C) Serum insulin levels 15 min after the injection of glucose to LKB1^lox/lox and PLKB mice (n = 7 mice per genotype). Mice were injected with tamoxifen at 4 weeks of age, and assay was performed 1 month later. Error bars represent standard deviations. **p < 0.01.

(A) Increased activity of mTOR pathway in islets isolated from PLKB mice. Western blots show decreased LKB1, decreased phosphorylation of AMPK and its target ACC, and increased phosphorylation of rpS6 and P70S6K.
(B) Low dose of rapamycin for 2 weeks reduces β cell size and eliminates the difference between LKB1^lox/lox and PLKB β cells. Mice were injected with tamoxifen at 5 weeks of age and sacrificed 3 weeks later (n = 2–3 mice per group).
(C) Genetic alterations in PI3K signaling pathway, but not Par1b deficiency, affect β cell size. For each mutant or transgenic, a coprocessed littermate is shown on the left. In (B) and (C), red is E-cadherin, and green is nuclei. Cell size was calculated from the circumference of individual cells. In the graphs, blue is wild-type, and red is transgenic or mutant. Original magnification in (B) and (C), 600×.
(D) Left, infection of Min6 cells with a lentivirus containing a dominant negative (DN) AMPK construct results in increased cell size, compared with cells infected with an EGFP-containing virus alone, 3 days after infection. Forward-scatter FACS plot of infected (EGFP⁺) and uninfected cells. Right, decreased phosphorylation of ACC in Min6 cells overexpressing DN AMPK. Metformin was added in the last 12 hr to maximize ACC phosphorylation.

(A) Insulin secretion in perfused pancreata, and the effect of rapamycin. Glucose was infused at 2.8 mM (G2.8) or 16.7 mM (G16.7) at the indicated times. Where indicated, mice were treated with rapamycin for 2 weeks prior to perfusion as described in Experimental Procedures. Data represent the mean ± SE of 4–6 mice. Mice were injected with tamoxifen at 4 weeks of age, and the assay was performed 2–3 months later. PLKB versus wild-type (no rapamycin), all time points between 11 and 27 min, p < 0.05. Wild-type treated with rapamycin, only t = 11 min has p < 0.05 compared with untreated wild-type. In PLKB mice, effect of rapamycin treatment was not significant at any time point. In this panel only, error bars denote standard errors.
(B) Blood glucose levels in Par1b^−/− mice and wild-type littermates, following the injection of glucose.
(C) Serum insulin levels in Par1b^−/− mice and wild-type littermates, following the injection of glucose. Graphs in (B) and (C) show data from females only. Males produced more variable results (not shown) with no difference between wild-type and mutants. Error bars in (B) and (C) represent standard deviation. N = 3 wild-type, 4 Par1b^−/− mice. Mice were 2–4 months old. Differences between wild-type and mutant in (B) and (C) were nonsignificant.
(D) Insulin secretion in response to low and high glucose in cultured PLKB islets.
(E) Insulin secretion in Min6 cells overexpressing wild-type or dominant negative LKB1.
(F) Insulin content in Min6 cells expressing wild-type or dominant negative LKB1 under low and high glucose. Cells were cultured overnight in 2.5 mM glucose, incubated in low glucose/no serum (KRB medium) for 2 hr, and then transferred to low or high glucose for 2 hr before being harvested for ELISA assay.
(G) Insulin secretion in Min6 cells overexpressing dominant negative AMPK or EGFP control.
(H) Insulin content in Min6 cells expressing EGFP or EGFP plus dominant negative AMPK. Experimental protocol was as in (F). Insulin content and secretion in (D)–(H) are expressed in ng insulin/μg protein.

(A) Immunofluorescence analysis of LKB1 expression in the adult pancreas. All islet cells express LKB1.
(B) Normal expression of LKB1 in adult pdx1-CreER™; LKB1^lox/lox (PLKB) islets in the absence of tamoxifen.
(C) Complete elimination of LKB1 immunoreactivity in β cells 7 days following tamoxifen injection to 2-month-old mice. Remaining LKB1⁺ islet cells are insulin⁻, probably representing alpha and PP cells that do not express pdx1 and therefore do not undergo recombination. All panels, original magnification 400×.

(A) Increased β cell size in LKB1-deficient mice, 3 months after the administration of tamoxifen to 6-week-old mice. Red, E-Cadherin; green, nuclei. Note also the abnormal clustering of nuclei around capillaries (yellow, autofluorescence of red blood cells). Islets are marked with dashed lines. Original magnification, 600×. Right, quantification of cell size. Top panel, distribution of islet cell sizes calculated from E-Cadherin immunostaining on paraffin sections. Each curve represents one mouse. All 6 mice depicted were littermates. Lower panel, FACS plot of dissociated islets from LKB1^lox/lox and PLKB mice. Blue, LKB1^lox/lox; red, PLKB. Right shift of mutant cells in both assays represents an increase in average cell size. PLKB = Pdx1-CreER; LKB1 lox/lox.
(B) Altered position of the β cell nucleus versus islet capillaries in PLKB mice. In both strains, β cells are organized as rosettes around capillaries (white arrows). In PLKB β cells, nuclei shift toward the center of rosettes. Red, E-Cadherin; blue, Laminin; green, nuclei. Right panels, quantification of distance of β cell nuclei from the center of rosettes (0 = center of rosette, 1 = farthest β cell membrane). Top, each bar represents nuclei from an individual rosette. Bottom, a graphic summary of data from 7 rosettes taken from 2 mice of each genotype. Blue, LKB1^lox/lox; red, PLKB. p < 0.01. Analysis was performed on same mice as in (A).
(C) Altered distribution of monocilia in PLKB islets. In LKB1^lox/lox islets, cilia are evenly spread and are usually located on lateral surfaces. In PLKB islets, cilia (arrowheads) are clustered in positions distant from the center of rosettes (arrows). Red, acetylated alpha tubulin; blue, Glut2; green, nuclei. Original magnification, 600×. Analysis was performed on same mice as in (A). Error bars denote standard deviations.

(A) Images of islets from several mutant strains, highlighting rosettes of β cells surrounding capillaries. The position of the nucleus in β cells (right panels) was determined relative to the center of rosettes. Red, E-Cadherin; blue, Laminin; green, nuclei. White lines denote rosettes. Blue, wild-type; red, mutant or transgenic. Original magnification, 600×. Analysis was performed on the same mice as in Figure 4.
(B) Nuclei are shifted to the center of rosettes in Par1b^−/− islets. Top, immunofluorescent image (magnification 600×) of representative rosettes from wild-type and Par1b^−/− islets, and quantification. Bottom, hematoxylin and eosin staining (400× magnification) showing abnormal distribution of nuclei in Par1b mutant islets.
(C) Reduced phosphorylation of Mark proteins in lysates from PLKB islets, compared with control LKB1^lox/lox islets. The upper band represents phosphorylated Mark3, lower band represents Mark2/Par1b. Error bars denote standard deviations.

(A) A model of β cell polarity and the role of LKB1 and Mark proteins in shifting cells from a default state of columnar polarity to hepatic-like polarity, characterized by a lateral lumen. Islet capillaries at the center of rosettes provide basement membrane proteins and define the basal side of β cells. Blue, basement membrane; green, nuclei; red, cilia.
(B) Proposed pathways for the impact of LKB1 on multiple features of β cells. Dashed lines represent pathways suggested but not proven by our data.