Table 1.
Primers used for RT-qPCR.
Figure 1.
Whole section imaging and analysis of mutant and wild-type mouse pancreases.
Pancreas sections from neuroligin-2 (NL-2) knockout (KO) and wild-type (WT) mice were stained for insulin (green), glucagon (red) and, as a nuclear counterstain, with DAPI (blue). A,B Representative whole-section images of stained pancreases: WT (A) and KO (B). C,D, Close-up of wild-type (C) and NL-2 KO (D) islets. E, Proportion of wild-type and neuroligin-2 knockout islets with alpha cells that appear to be in the beta-cell core region. F, Average beta-cell area per islet, shown relative to wild-type value. G, Islet density was measured by dividing the number of islets in each section by the total pancreas area in the section. Results are shown relative to wild-type values H, Total insulin area as a percentage of total pancreas section area, shown as a percentage of the wild-type value. All data are represented as mean +/− SEM (n = 8 sections at 80 µm intervals from each of 3 KO and 3 paired WT pancreases taken from 3–4 month old female littermates). ***represents a p<0.001.
Figure 2.
Islet and total pancreatic insulin content.
Islets were isolated from NL-2 knockout (KO) and wild-type (WT) mice (white bars, WT; black bars, KO). A, Similarly-sized islets were hand-picked, lysed, and then insulin content was measured by RIA. B, Average protein content of picked islets. C, Islet insulin content normalized to total islet protein content. D, Insulin content in intact pancreases. Insulin content (ng) is shown normalized to weight (in mg) of pancreatic tissue analyzed. All data is represented as mean +/− SEM (n = 3 littermate pairs of KO and WT mice). ***represents a p<0.001; *represents p<0.05.
Figure 3.
Insulin secretion by islets from mutant and wild-type mice.
Islets were isolated from neurolgin-2 knockout (KO) and wild-type (WT) mice and allowed to recover overnight in culture. A, insulin secretion by islets in static culture. Islets from KO (black columns) and WT (white columns) mice were incubated for 1 h with either 2.75 mM (low) or 16.7 mM (high) glucose. Insulin secreted as a percentage of total islet insulin content is shown. Results are representative of three independent experiments. B,C, Insulin secretion by perifused islets. Islets were first perifused in buffer with 2.5 mM glucose for 30 min, with samples being collected during the last 10 min for analysis. Glucose was raised to 20 mM and perifusion was continued for another 40 min. Next, during the last 40 min, islets were perifused with 20 mM glucose and GLP-1 (100 nM). Insulin secretion was assessed as mean integrated area under the curve (AUC) of insulin secretory rate (ISR). In B, the relative average areas under the curve of the insulin secretory rate for first-phase insulin secretion (2 min through 8 min after stimulation with 20 mM glucose), for second-phase insulin secretion (from end of first phase up until addition of GLP-1) and for GLP-1-stimulated insulin secretion (first time point after addition of GLP-1 through end of perifusion) are shown. AUCs from KO islets (black columns) are shown relative to average AUCs from perifusions with WT islets (white columns). In C, the average ISR for each time point beginning with the time point 10 min prior to stimulation with 20 mM glucose is depicted. The glucose concentrations in the perifusion medium are shown above the graph, as is the time interval during which GLP-1 was also added. The ISR is the percentage of islet insulin content released per minute; the y axis shows ISR multiplied by 10 (n = 5 experiments, each with islets from pairs of KO and WT mice perifused in parallel). ***p<0.001; **, p<0.01; *p<0.05.
Figure 4.
Insulin secretion after knockdown of neuroligin-2 in cultured islets.
Rat islets were infected with adenoviruses expressing either shRNA against neuroligin-2 (NL2) or a control shRNA construct (Cav3) or were left uninfected (untreated). Adenovirus-containing media was removed after 24 h, and islets were cultured an additional 6 days prior to assessment of insulin secretion. A, Insulin secretion by islets that were either untreated or infected with adenovirus expressing shRNA was assessed after 1 h incubation in either 2.75 mM (low) or 16.7 mM (high) glucose. Insulin secreted as a percentage of total islet insulin content is shown. Results are representative of three independent experiments. B, NL2 message levels were assessed by real-time qPCR at the end of the culture period. Transcript levels are shown relative to the level in untreated islets. *p<0.05; **p<0.01; ***p<0.001.
Figure 5.
Decreased neurexin and increased neuroligin-1 transcript levels in KO islets.
Real-time qPCR was used to analyze expression of neurexin-1α (NRXN 1a) and neurexin-2β (NRXN 2b); also of two representative constituents of the insulin exocytic machinery, the voltage-gated calcium channel CaV1.3 (VGCC) and Munc18; and also of the inhibitory synaptic protein GAD67; and also the expression of neuroligin-1 (NL1). Transcript levels were assessed in islets from knockout (KO; black columns) and wild-type (WT; white columns) mice. Transcript levels are shown relative to levels in the WT mice (n = 3 mice of each genotype). ***p<0.001; **p<0.01.
Figure 6.
Analysis of insulin granule docking in mutant and wild-type beta cells.
After a 24 h recovery period in culture, islets isolated from NL-2 KO and WT mice were fixed, pelleted and sectioned for EM. A, Representative electron micrographs of WT(top) and NL-2 KO (bottom) beta cells. Cell border is traced in solid black. Dashed lines are drawn 200 nM and 400 nM from the plasma membrane. Docked granules, those with centers located from 100 nm to 200 nm from the plasma membrane, are indicated by arrows. B, Distance from the center of each granule to the plasma membrane was measured and separated into four groups. The numbers in each group were then normalized to the total length of plasma membrane of the cell in question to determine linear density (granules per 1000 nm). Investigators were blinded to mouse identity during EM image analysis. All data are represented as mean +/− SEM from 20 cells. **, p<0.01.