Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of \u3e95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days

Deregulated lipid sensing by intestinal CD36 in diet-induced hyperinsulinemic obese mouse model

The metabolic syndrome (MetS) greatly increases risk of cardiovascular disease and diabetes and is generally associated with abnormally elevated postprandial triglyceride levels. We evaluated intestinal synthesis of triglyceride-rich lipoproteins (TRL) in a mouse model of the MetS obtained by feeding a palm oil-rich high fat diet (HFD). By contrast to control mice, MetS mice secreted two populations of TRL. If the smaller size population represented 44% of total particles in the beginning of intestinal lipid absorption in MetS mice, it accounted for only 17% after 4 h due to the secretion of larger size TRL. The MetS mice displayed accentuated postprandial hypertriglyceridemia up to 3 h due to a defective TRL clearance. These alterations reflected a delay in lipid induction of genes for key proteins of TRL formation (MTP, L-FABP) and blood clearance (ApoC2). These abnormalities associated with blunted lipid sensing by CD36, which is normally required to optimize jejunal formation of large TRL. In MetS mice CD36 was not downregulated by lipid in contrast to control mice. Treatment of controls with the proteosomal inhibitor MG132, which prevented CD36 downregulation, resulted in blunted lipid-induction of MTP, L-FABP and ApoC2 gene expression, as in MetS mice. Absence of CD36 sensing was due to the hyperinsulinemia in MetS mice. Acute insulin treatment of controls before lipid administration abolished CD36 downregulation, lipid-induction of TRL genes and reduced postprandial triglycerides (TG), while streptozotocin-treatment of MetS mice restored lipid-induced CD36 degradation and TG secretion. In vitro, insulin treatment abolished CD36-mediated up-regulation of MTP in Caco-2 cells. In conclusion, HFD treatment impairs TRL formation in early stage of lipid absorption via insulin-mediated inhibition of CD36 lipid sensing. This impairment results in production of smaller TRL that are cleared slowly from the circulation, which might contribute to the reported association of CD36 variants with MetS risk

Gastrin Induces Nuclear Export and Proteasomal Degradation of Menin in Enteric Glial Cells

Background & aims: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner\u27s glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Methods: Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Results: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. Conclusions: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae

Coordinate induction of genes involved in chylomicron synthesis and clearance is delayed in MetS mice.

<p>After overnight fasting, control and MetS mice were gavaged with 0.5 mL oil and sacrificed 1 and 6 h later. Jejunal mRNA levels of genes of interest were evaluated by real-time PCR and normalized to 36B4 mRNA. Induction of gene expression is shown at 1 h (A) and 6 h (B) after the lipid load as compared with fasting in control and MetS mice. Means ± SEM, n = 5 or 6, *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001. Induction of gene expression obtained from control and MetS mice was also compared across groups # <i>P</i> < 0.05, ## <i>P</i> < 0.01.</p

Lipid-mediated induction of genes involved in chylomicron synthesis is dependent on CD36 proteasomal degradation.

<p>Fasted CD36 (+/+) and CD36 (-/-) mice were intraperitoneally injected with MG132 (14 mg/kg) 30 min before oil gavage and sacrificed 4 h later. (A) CD36 expression standardized to HSC70 as the loading control. (B) mRNA level analysis by real-time PCR normalized to 36B4 mRNA. Means ± SEM, n = 5. *<i>P</i> < 0.05.</p

MetS mice display higher postprandial lipemia than controls and altered TRL particle distribution.

<p>Plasma TG (A) and NEFA (B) quantified after an intragastric lipid load (0.5 mL) in control and MetS mice. TG secretion as a function of time after or not the lipid load in mice pre-injected retro-orbitally with the LPL inhibitor (tyloxapol, 500 mg/kg) (C). Lipoprotein particle distribution in the plasma estimated using DLS in mice injected with tyloxapol at 1h (D), 2 h (E) and 4 h (F) after the lipid bolus. Means ± SEM, n = 8 (A, B and C), n = 5 (D, E), n = 11 (F), means with same letter are not significantly different, *<i>P</i> < 0.05, ***<i>P</i> < 0.001.</p

Streptozotocin treatment restores lipid-downregulation of CD36 and its associated effects in MetS mice.

<p>A retro-orbital injection of streptozotocin (100 mg/kg) was performed in fasted MetS mice. Six days later, fasted MetS mice were treated with tyloxapol (500mg/kg), given a lipid-bolus and sacrificed 1.5 h later. (A) CD36 expression in jejunal mucosa (B) Streptozotocin effect on mRNA levels of key chylomicron genes by real-time PCR with data normalized to 36B4 mRNA (C). Plasma TG secretion. Means ± SEM, n = 5, *P<0.05.</p