LKB1 deletion with the RIP2.Cre transgene modifies pancreatic beta-cell morphology and enhances insulin secretion in vivo

Abstract

The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic beta-cell and hypothalamic neurons using the rat insulin promoter (RIP2).Cre transgene (betaAMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the beta-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the beta-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to betaAMPKdKO mice, betaLKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1-CreER mice, total beta-cell mass and the size of individual islets and beta-cells were increased and islet architecture was markedly altered in betaLKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular Ca(2+) were sharply reduced in betaLKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on beta-cell function.

Fig. 1.

Generation of βLKB1KO mice. A: schematic representation of deletion of flox’d lkb1 exons (exons 3–6) driven by RIP2.Cre expression. Location of primers used for PCR as indicated. Black arrows, flox sites; gray bar, LKB1 exons. B: RT-PCR analysis of effects on LKB1 transcript levels of deleting exons 3–6 in pancreatic islets and hypothalamus (Hypo). Product sizes were 864 and 300 bp for flox’d and null alleles, respectively. NS, nonspecific. C: qRT-PCR (C) and Western blot analysis (D) of LKB1 mRNA and protein levels in pancreatic islets of βLKB1KO mice and their heterozygous (het) and wild-type (Wt) controls. Total AMPK activity in islets (E) and hypothalamus (F) of βLKB1KO and βLKB1het mice. Data are expressed as means ± SE; n = 4–5 mice per genotype. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Food intake and glucose homeostasis in βLKB1KO mice. A–C: body weight (A), and food intake of βLKB1KO mice fed (B) or refed after fasting for 15 h (C). D–F: blood glucose (D and E) and plasma insulin (F) of βLKB1KO mice fed or fasted for 15 h. Male mice from 6–8 wk old were used. Data are expressed as means ± SE; *P < 0.05, **P < 0.01; n = 7–10 mice per genotype.

Fig. 3.

Glucose and insulin tolerance of βLKB1KO mice. Glucose tolerance (A), and plasma insulin response (B) after intraperitoneal (ip) glucose injection, and whole body insulin sensitivity (C) monitored after ip insulin injection of βLKB1KO or control mice. Male 6- to 8-wk-old mice old were used. Data are expressed as means ± SE. For A and B, *P < 0.05, **P < 0.01 for βLKB1KO vs. βLKB1Wt, ⁺P < 0.05 for βLKB1KO vs. βLKB1het; in C, *P < 0.05 with respect to time 0 for all genotypes; n = 7–10 mice per genotype.

Fig. 4.

Altered islet morphology in βLKB1KO mice. A: representative optical projection tomographic (OPT) images of whole pancreas. OPT was performed as described under methods. Red staining indicates insulin-positive structures (islets), while the outline of the whole pancreas was apparent as autofluorescence and is presented as white/gray shading. Images shown correspond to 3-D projections; (see also Suppl. Fig. 2 and Suppl. Movies betaLKB1wt, betaLKB1het, and betaLKB1KO.avi.) Note presence of a particularly large islet in the βLKB1KO pancreas (right image, arrow). B: distribution of islet volumes with marked (dotted lines) section magnified. C: relative β-cell mass; D: mean islet volume. Data are from 5–6 mice per genotype. Scale bar, 500 μm. E: hematoxylin-eosin (H&E) and immunofluorescent staining of pancreatic sections using guinea pig anti-insulin (1:200; green) and rabbit anti-glucagon (1:100; red) antibodies. Nuclei are shown with DAPI (blue) staining. Scale bar, 75 μm. F: representative E-cadherin staining of pancreatic sections and quanification of single islet β-cell sizes. Average area of 100 single β-cells from 5 islets of each genotype, costained in pancreatic sections for E-cadherin and insulin, were analyzed. Scale bar, 12 μm. G: staining for proliferation marker Ki67 and quantification, based on 15–20 islets per pancreas; n = 3 mice per genotype. H: Western blot analysis of mTOR signaling pathway markers phospho-ribosomal protein (rp)S6 and phospho-4E-BP1 of pancreatic islet extracts from βLKB1KO and control mice. Islets extracted from fed mice were incubated in RPMI supplemented with 11 mM glucose for 16 h and lysed for analysis. Data are expressed as means ± SE. *P < 0.05, **P < 0.01. In B, *P < 0.05, for βLKB1KO vs. βLKB1Wt, $P < 0.05 for βLKB1KO vs. βLKB1het.

Fig. 5.

β-Cell polarity is enhanced in βLKB1KO mice with increased rosette-like (“islet acini”) structures and accumulation of cell junction proteins. A–E: immunofluorescence staining of pancreatic sections for the adherens junction marker (A and B) E-cadherin (1:100, green), (C) actin filament (1:50, red; note imaged areas were confirmed as lying over an islet by inspection of corresponding bright field images), (D) β-tubulin (1:100, green), (E) tight junction marker zona occludins 1 (ZO1, whole serum; green) or capillary marker (F) VEGFR2 (1:100, green). White square, voids at center of rosettes. G: quantification of no. of rosette-like structures per islet in βLKB1KO mice based on E-cadherin and DAPI staining. Such structure in islet is counted as 1 by E-cadherin staining where the voids at the center of the rosette is absent of DAPI staining. Ten islets from 3 pairs of mice per genotype were assessed. Data are expressed as means ± SE. **P < 0.01, ***P < 0.001. Scale bars, 50 μm (A), 12 μm (B), 10 μm (C), and 25 μm (D–F).

Fig. 6.

βLKB1KO β-cells display abnormal electrical, Ca²⁺, and secretory responses and decreased GLUT2 immunoreactivity at the plasma membrane. A: glucose-stimulated insulin secretion and total insulin content of 6 size-matched islets statically incubated with indicated glucose for 0.5 h from βLKB1KO, heterozygous, and WT mice; n = 3 mice per genotype. B: representative traces of whole cell K_ATP channel conductance (G_KATP) and plasma membrane potential (V_m) from perforated patch clamp measurements. Three to six β-cells from 3 pairs of mice of each genotype were recorded. C: representative traces and quantification of free [Ca²⁺] with fura 2-AM in dissociated β-cells in βLKB1KO mice. Cells (38–60) from 3 pairs of mice of each genotype were analyzed. D: immunofluorescence staining and quantification of Glut2 expression at the plasma membrane of pancreatic islet β-cells. Fifteen islets from 2 pairs of mice per genotype were examined. Scale bar, 75 μm. Data are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

In vivo metabolic advantages of LKB1 deletion from insulin-expressing cells are lost on a high-fat diet. Glucose tolerance (A) and plasma insulin changes (B) after ip glucose injection of βLKB1KO mice on a high-fat diet for 6 wk (see methods).