Xinping Lu

The G-protein-coupled receptor GPR40 directly mediates long-chain fatty acid-induced secretion of cholecystokinin.

BACKGROUND & AIMS Long-chain fatty acid receptors G-protein-coupled receptor 40 (GPR40) (FFAR1) and GPR120 have been implicated in the chemosensation of dietary fats. I cells in the intestine secrete cholecystokinin (CCK), a peptide hormone that stimulates digestion of fat and protein, but these cells are rare and hard to identify. We sought to determine whether dietary fat-induced secretion of CCK is directly mediated by GPR40 expressed on I cells. METHODS We used fluorescence-activated cell sorting to isolate a pure population of I cells from duodenal mucosa in transgenic mice that expressed green fluorescent protein under the control of the CCK promoter (CCK-enhanced green fluorescent protein [eGFP] bacterial artificial chromosome mice). CCK-eGFP cells were evaluated for GPR40 expression by quantitative reverse transcription polymerase chain reaction and immunostaining. GPR40(-/-) mice were bred with CCK-eGFP mice to evaluate functional relevance of GPR40 on long-chain fatty acid-stimulated increases in [Ca(2+)]i and CCK secretion in isolated CCK-eGFP cells. Plasma levels of CCK after olive oil gavage were compared between GPR40(+/+) and GPR40(-/-) mice. RESULTS Cells that expressed eGFP also expressed GPR40; expression of GPR40 was 100-fold greater than that of cells that did not express eGFP. In vitro, linoleic, oleic, and linolenic acids increased [Ca(2+)]i; linolenic acid increased CCK secretion by 53% in isolated GPR40(+/+) cells that expressed eGFP. In contrast, in GPR40(-/-) that expressed eGFP, [Ca(2+)]i response to linoleic acid was reduced by 50% and there was no significant CCK secretion in response to linolenic acid. In mice, olive oil gavage significantly increased plasma levels of CCK compared with pregavage levels: 5.7-fold in GPR40(+/+) mice and 3.1-fold in GPR40(-/-) mice. CONCLUSIONS Long-chain fatty acid receptor GPR40 induces secretion of CCK by I cells in response to dietary fat.

The extracellular calcium sensing receptor is required for cholecystokinin secretion in response to L-phenylalanine in acutely isolated intestinal I cells

The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an L-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of L-phenylalanine (L-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for L-Phe over D-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to L-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of L-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca(2+), evoked an unexpected 20-30% decrease in CCK secretion compared with basal secretion in CaSR(-/-) CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to L-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR.

Postprandial inhibition of gastric ghrelin secretion by long-chain fatty acid through GPR120 in isolated gastric ghrelin cells and mice

Ghrelin is a gastric peptide hormone that controls appetite and energy homeostasis. Plasma ghrelin levels rise before a meal and fall quickly thereafter. Elucidation of the regulation of ghrelin secretion has been hampered by the difficulty of directly interrogating ghrelin cells diffusely scattered within the complex gastric mucosa. Therefore, we generated transgenic mice with ghrelin cell expression of green fluorescent protein (GFP) to enable characterization of ghrelin secretion in a pure population of isolated gastric ghrelin-expressing GFP (Ghr-GFP) cells. Using quantitative RT-PCR and immunofluorescence staining, we detected a high level of expression of the long-chain fatty acid (LCFA) receptor GPR120, while the other LCFA receptor, GPR40, was undetectable. In short-term-cultured pure Ghr-GFP cells, the LCFAs docosadienoic acid, linolenic acid, and palmitoleic acid significantly suppressed ghrelin secretion. The physiological mechanism of LCFA inhibition on ghrelin secretion was studied in mice. Serum ghrelin levels were transiently suppressed after gastric gavage of LCFA-rich lipid in mice with pylorus ligation, indicating that the ghrelin cell may directly sense increased gastric LCFA derived from ingested intraluminal lipids. Meal-induced increase in gastric mucosal LCFA was assessed by measuring the transcripts of markers for tissue uptake of LCFA, lipoprotein lipase (LPL), fatty acid translocase (CD36), glycosylphosphatidylinositol-anchored HDL-binding protein 1, and nuclear fatty acid receptor peroxisome proliferator-activated receptor-γ. Quantitative RT-PCR studies indicate significantly increased mRNA levels of lipoprotein lipase, glycosylphosphatidylinositol-anchored HDL-binding protein 1, and peroxisome proliferator-activated receptor-γ in postprandial gastric mucosa. These results suggest that meal-related increases in gastric mucosal LCFA interact with GPR120 on ghrelin cells to inhibit ghrelin secretion.

A stem cell marker-expressing subset of enteroendocrine cells resides at the crypt base in the small intestine

The spatial orientation of the enteroendocrine cells along the crypt-villus axis is closely associated with their differentiation in the intestine. Here we studied this relationship using primary duodenal crypts and an ex vivo organoid system established from cholecystokinin-green fluorescent protein (CCK-GFP) transgenic mice. In the primary duodenal crypts, GFP+ cells were found not only in the upper crypt but also at the crypt base, where the stem cells reside. Many GFP+ cells below +4 position were positive for the putative intestinal stem cell markers, leucine-rich repeat-containing G protein-coupled receptor 5, CD133, and doublecortin and CaM kinase-like-1, and also for the neuroendocrine transcription factor neurogenin 3. However, these cells were neither stem nor transient amplifying precursor cells because they were negative for both Ki-67 and phospho-Histone H3 and positive for the mature endocrine marker chromogranin A. Furthermore, these cells expressed multiple endocrine hormones. Tracking of GFP+ cells in the organoids from CCK-GFP mice indicated that GFP+ cells were first observed around the +4 position, some of which localized to the crypt base later in the culture period. These results suggest that a subset of enteroendocrine cells migrates down to the crypt base or stays localized at the crypt base, where they express stem and postmitotic endocrine markers. Further investigation of the function of this subset may provide novel insights into the genesis and development of enteroendocrine cells as well as enteroendocrine tumorigenesis.

Synthesis, Receptor Binding, and CNS Pharmacological Studies of New Thyrotropin-Releasing Hormone (TRH) Analogues

As part of our search for selective and CNS‐active thyrotropin‐releasing hormone (TRH) analogues, we synthesized a set of 44 new analogues in which His and pGlu residues were modified or replaced. The analogues were evaluated as agonists at TRH‐R1 and TRH‐R2 in cells in vitro, and in vivo in mice for analeptic and anticonvulsant activities. Several analogues bound to TRH‐R1 and TRH‐R2 with good to moderate affinities, and are full agonists at both receptor subtypes. Specifically, analogue 21 a (R=CH3) exhibited binding affinities (Ki values) of 0.17 μM for TRH‐R1 and 0.016 μM for TRH‐R2; it is 10‐fold less potent than TRH in binding to TRH‐R1 and equipotent with TRH in binding to TRH‐R2. Compound 21 a, the most selective agonist, activated TRH‐R2 with a potency (EC50 value) of 0.0021 μM, but activated TRH‐R1 at EC50=0.05 μM, and exhibited 24‐fold selectivity for TRH‐R2 over TRH‐R1. The newly synthesized TRH analogues were also evaluated in vivo to assess their potencies in antagonism of barbiturate‐induced sleeping time, and several analogues displayed potent analeptic activity. Specifically, analogues 21 a,b and 22 a,b decreased sleeping time by nearly 50 % more than TRH. These analogues also displayed potent anticonvulsant activity and provided significant protection against PTZ‐induced seizures, but failed to provide any protection in MES‐induced seizures at 10 μmol kg−1. The results of this study provide evidence that TRH analogues that show selectivity for TRH‐R2 over TRH‐R1 possess potent CNS activity.