Carbonic anhydrase 8 (CAR8) negatively regulates GLP-1 secretion from enteroendocrine cells in response to long-chain fatty acids.

Glucagon-like peptide-1 (GLP-1) is an incretin secreted from enteroendocrine preproglucagon (PPG)-expressing cells (traditionally known as L cells) in response to luminal nutrients that potentiates insulin secretion. Augmentation of endogenous GLP-1 secretion might well represent a novel therapeutic target for diabetes treatment in addition to the incretin-associated drugs currently in use. In this study, we found that PPG cells substantially express carbonic anhydrase 8 (CAR8), which has been reported to inhibit inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor and subsequent Ca2+ efflux from the endoplasmic reticulum in neuronal cells. In vitro experiments using STC-1 cells demonstrated that Car8 knockdown increases long-chain fatty acid (LCFA)-stimulated GLP-1 secretion. This effect was reduced in the presence of phospholipase C (PLC) inhibitor; in addition, Car8 knockdown increased the intracellular Ca2+ elevation caused by α-linolenic acid, indicating that CAR8 exerts its effect on GLP-1 secretion via the PLC/IP3/Ca2+ pathway. Car8wdl null mutant mice showed significant increase in GLP-1 response to oral corn oil administration compared with that in wild-type littermates, with no significant change in intestinal GLP-1 content. These results demonstrate that CAR8 negatively regulates GLP-1 secretion from PPG cells in response to LCFAs, suggesting the possibility of augmentation of postprandial GLP-1 secretion by CAR8 inhibition.NEW & NOTEWORTHY This study focused on the physiological significance of carbonic anhydrase 8 (CAR8) in GLP-1 secretion from enteroendocrine preproglucagon (PPG)-expressing cells. We found an inhibitory role of CAR8 in LCFA-induced GLP-1 secretion in vitro and in vivo, suggesting a novel therapeutic approach to diabetes and obesity through augmentation of postprandial GLP-1 secretion by CAR8 inhibition.

Silychristin A activates Nrf2-HO-1/SOD2 pathway to reduce apoptosis and improve GLP-1 production through upregulation of estrogen receptor α in GLUTag cells.

Glucagon-like peptide-1 (GLP-1), a glucagon-like peptide secreted mainly from intestinal L cells, possesses the functions of promoting synthesis and secretion of insulin in pancreatic ß-cells, and maintaining glucose homeostasis in an insulin-independent manner. Silychristin A, a major flavonolignan from silymarin, was reported to protect pancreatic ß-cells from oxidative damage in streptozotocin (STZ)-induced diabetic rats. However, the role of silychristin A in the protection of intestinal L-cells is still unknown. Our current study demonstrated that palmitate (PA) inhibited protein expression of NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and superoxide dismutase 2 (SOD2), and subsequently increased reactive oxygen species level to induce apoptosis and decrease GLP-1 content in intestinal L-cell line GLUTag cells. Pre-incubation of silychristin A effectively reversed PA-inactivated Nrf2-HO-1/SOD2 antioxidative pathway accompanied with decreased apoptosis level and increased GLP-1 level in GLUTag cells. As a potential target of silychristin A, estrogen receptor α was shown to be downregulated by PA stimulation, and the expression of which was improved by silychristin A in a concentration-dependent manner. Further study revealed that the treatment of estrogen receptor α antagonist MPP induced apoptosis and blocked the stimulation of GLP-1 production by silychristin A through the activation of Nrf2-HO-1/SOD2 pathway in GLUTag cells. Taken together, our study found silychristin A activated estrogen receptor α-dependent Nrf2-HO-1/SOD2 pathway to decrease apoptosis and upregulate GLP-1 production in GLUTag cells.

Glutamine-Elicited Secretion of Glucagon-Like Peptide 1 Is Governed by an Activated Glutamate Dehydrogenase.

Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from ß-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo.

Alternative form of glucose-dependent insulinotropic polypepide and its physiology.

Glucose-dependent insulinotropic polypepide (GIP) was first extracted from porcine gut mucosa and identified as "incretin" decades ago. Though early studies have shown the possible GIP isoforms by gel filtration profiles from porcine or human intestinal extracts analyzed by radioimmunoassay (RIA), GIP is currently believed to consist of 42 amino acids (GIP1-42), which are released from gut K-cells and promote postprandial insulin release. In fact, GIP1-42 is usually processed from proGIP by the action of prohormone convertase (PC) 1/3 in the gut. GIP expression is occasionally found in the intestinal glucagon-like peptide-1-secreting cells, suggesting gene expression of both GIP and proglucagon can co-exist in identical cells. However, GIP1-42 immunoreactivity is rarely found in α-cells or other pancreatic endocrine cells of wild-type mammals. Interestingly, we found that short-form GIP1-30 is expressed in and released from pancreatic α-cells and a subset of enteroendocrine cells through proGIP processing by PC2. GIP1-30 is also insulinotropic and modulates glucose-stimulated insulin secretion in a paracrine manner. It is also suggested that short-form GIP1-30 possibly plays a crucial role for the islet development. It has not been well elucidated whether expression of GIP1-30 is modulated in the diabetic status, and whether GIP1-30 might have therapeutic potentials. Our preliminary data suggest that short-form GIP1-30 might play important roles in glucose metabolism.

Enteroendocrine cells are a potential source of serum autotaxin in men.

OBJECTIVE: Serum autotaxin (ATX) activity is significantly increased in cholestatic patients. Our study aimed to unravel the source(s) of ATX in cholestasis. MATERIALS AND METHODS: ATX activity and protein were measured in sera of healthy (n=33) and cholestatic patients (n=152), including women with intrahepatic cholestasis of pregnancy. ATX mRNA and protein expression were analyzed in various tissues from mice and men. Induction of ATX activity was assessed in mouse models of extrahepatic (bile duct ligation) and intrahepatic cholestasis (Atp8b1(G308V/G308V), 0.1% cholate-supplemented diet). ATX clearance in cholestatic and control mice was assessed after intravenous injection of recombinant ATX. Human hepatic clearance was estimated by comparing ATX activity in portal and hepatic vein serum. RESULTS: Serum ATX activity and ATX protein concentration tightly correlated under all conditions in patients and controls (p<0.0001). In humans Atx mRNA was highly expressed in small intestine, whereas in mice Atx was expressed mainly in brain and placenta but not in small intestine. Extensive ATX protein expression was identified in human, but not murine intestinal enteroendocrine cells. In murine models of cholestasis and cholestatic pregnancy plasma ATX activity was only mildly elevated (up to 2.1-fold). Atx tissue expression and rATX clearance after parenteral administration did not differ between cholestatic and control mice. CONCLUSION: Serum ATX activity during cholestasis and itch is enhanced by increased protein concentration rather than enzymatic induction. In mice, clearance of ATX is not affected by cholestasis. Small intestinal ATX expression by enteroendocrine cells might represent an important source of cholestasis-induced serum ATX activity in men.

ADAM10 regulates Notch function in intestinal stem cells of mice.

BACKGROUND & AIMS: A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a cell surface sheddase that regulates physiologic processes, including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types, but the requirement for ADAM10 signaling in crypt homeostasis is not well defined. METHODS: We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10(f/f) mice) and conditional (Vil-CreER;Adam10(f/f) and Leucine-rich repeat-containing GPCR5 [Lgr5]-CreER;Adam10(f/f) mice) deletion of ADAM10. We performed cell lineage-tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26(NICD)), or mice with intestine-specific disruption of Notch (Rosa26(DN-MAML)), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance. RESULTS: Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26(NICD) and Rosa26(DN-MAML) mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage-tracing experiments showed that ADAM10 is required for survival of Lgr5(+) crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche. CONCLUSIONS: ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance.

The role of the PDE4D cAMP phosphodiesterase in the regulation of glucagon-like peptide-1 release.

BACKGROUND AND PURPOSE: Increases in intracellular cyclic AMP (cAMP) augment the release/secretion of glucagon-like peptide-1 (GLP-1). As cAMP is hydrolysed by cAMP phosphodiesterases (PDEs), we determined the role of PDEs and particularly PDE4 in regulating GLP-1 release. EXPERIMENTAL APPROACH: GLP-1 release, PDE expression and activity were investigated using rats and GLUTag cells, a GLP-1-releasing cell line. The effects of rolipram, a selective PDE4 inhibitor both in vivo and in vitro and stably overexpressed catalytically inactive PDE4D5 (D556A-PDE4D5) mutant in vitro on GLP-1 release were investigated. KEY RESULTS: Rolipram (1.5 mg x kg(-1) i.v.) increased plasma GLP-1 concentrations approximately twofold above controls in anaesthetized rats and enhanced glucose-induced GLP-1 release in GLUTag cells (EC(50) approximately 1.2 nmol x L(-1)). PDE4D mRNA transcript and protein were detected in GLUTag cells using RT-PCR with gene-specific primers and Western blotting with a specific PDE4D antibody respectively. Moreover, significant PDE activity was inhibited by rolipram in GLUTag cells. A GLUTag cell clone (C1) stably overexpressing the D556A-PDE4D5 mutant, exhibited elevated intracellular cAMP levels and increased basal and glucose-induced GLP-1 release compared with vector-transfected control cells. A role for intracellular cAMP/PKA in enhancing GLP-1 release in response to overexpression of D556A-PDE4D5 mutant was demonstrated by the finding that the PKA inhibitor H89 reduced both basal and glucose-induced GLP-1 release by 37% and 39%, respectively, from C1 GLUTag cells. CONCLUSIONS AND IMPLICATIONS: PDE4D may play an important role in regulating intracellular cAMP linked to the regulation of GLP-1 release.

[Clinical and morphological characteristics and some mechanisms of portal gastroduodenopathy in liver cirrhosis].

We have found out the clinical presentations and peculiarities of endoscopic and morphologic view of pathologies of mucous membrane of gastroduodenal zone caused by liver cirrhosis. We have examined 74 patients with liver cirrhosis of viral and nonviral etiology using the clinical, endoscopic, morphologic and immunohistochemical methods.We have found that during liver cirrhosis morphometric rates of epithelial cells of mucous coat of stomach that produce somatostatin and endothelin-1 decrease and morphometric rates of epithelial cells that produce nitrogen oxide synthase increase. We have also found out that during liver cirrhosis proliferate activity decrease and apoptosis of epithelial cell of mucous coat of stomach increase.

Mechanisms of action of glucagon-like peptide 1 in the pancreas.

Glucagon-like peptide 1 (GLP-1) is a hormone that is encoded in the proglucagon gene. It is mainly produced in enteroendocrine L cells of the gut and is secreted into the blood stream when food containing fat, protein hydrolysate, and/or glucose enters the duodenum. Its particular effects on insulin and glucagon secretion have generated a flurry of research activity over the past 20 years culminating in a naturally occurring GLP-1 receptor (GLP-1R) agonist, exendin 4 (Ex-4), now being used to treat type 2 diabetes mellitus (T2DM). GLP-1 engages a specific guanine nucleotide-binding protein (G-protein) coupled receptor (GPCR) that is present in tissues other than the pancreas (brain, kidney, lung, heart, and major blood vessels). The most widely studied cell activated by GLP-1 is the insulin-secreting beta cell where its defining action is augmentation of glucose-induced insulin secretion. Upon GLP-1R activation, adenylyl cyclase (AC) is activated and cAMP is generated, leading, in turn, to cAMP-dependent activation of second messenger pathways, such as the protein kinase A (PKA) and Epac pathways. As well as short-term effects of enhancing glucose-induced insulin secretion, continuous GLP-1R activation also increases insulin synthesis, beta cell proliferation, and neogenesis. Although these latter effects cannot be currently monitored in humans, there are substantial improvements in glucose tolerance and increases in both first phase and plateau phase insulin secretory responses in T2DM patients treated with Ex-4. This review will focus on the effects resulting from GLP-1R activation in the pancreas.

Nutrient-induced alpha-amylase and protease activity is regulated by crustacean cardioactive peptide (CCAP) in the cockroach midgut.

The midgut plays a major role in digestion and absorption of nutrients in insects, and contains endocrine cells throughout the epithelial layer that express neuropeptides, including crustacean cardioactive peptide (CCAP). In the present study, we demonstrate regulation of digestive enzyme activities by CCAP in response to nutrient ingestion in the cockroach, Periplaneta americana. The midgut of the cockroach exhibits maximal alpha-amylase and protease activities 3 h after intake of either starch or casein, but not of non-nutrients. Similar time-dependent responses of CCAP expression in midgut endocrine cells were observed after feeding starch and casein, but not after non-nutrients. We also show that incubation of the dissected midgut with CCAP leads to an increase in alpha-amylase and protease activity in a time-dependent manner, with the maximal activity at 2 h. Taken together, our data indicate the existence of an inducible mechanism where endocrine cells in the midgut are stimulated to synthesize and secrete CCAP by nutrients, and CCAP then up-regulates the activity of digestive enzymes.

Carboxypeptidase E in rat antropyloric mucosa: distribution in progenitor and mature endocrine cell types.

Processing of most gut hormones involves cleavage between dibasic amino acids followed by carboxypeptidase-catalyzed removal of the COOH-terminal basic residue, resulting in peptides with a COOH-terminal glycine. Such peptides may subsequently be converted to amidated peptides or can be directly secreted. It is believed that carboxypeptidase E (CPE) is involved in gut hormone processing but its presence in gut endocrine cells has never been studied. We have analyzed the distribution of CPE in the antropyloric mucosa of rat stomach and report that gastrin cells and progenitor gastrin-somatostatin (G/D) cells express CPE while mature somatostatin cells and the majority of serotonin cells fail to express CPE. These data indicate that immature G/D cells are able to process gastrin to glycine-extended forms and that CPE-mediated processing is not a characteristic of mature somatostatin and serotonin cells.

Gastric PDX-1 expression in pancreatic metaplasia and endocrine cell hyperplasia in atrophic corpus gastritis.

The homeodomain transcription factor PDX-1 plays a key role in endocrine and exocrine differentiation processes of the pancreas. PDX-1 is also essential for differentiation of endocrine cells in the gastric antrum. The role of PDX-1 in the pathogenesis of endocrine cell hyperplasia and pancreatic metaplasia in corpus and fundus gastritis has not been evaluated. By immunohistochemistry and double-immunofluorescence, we investigated the expression of PDX-1 in 10 tissue specimens with normal human gastric mucosa, nonatrophic and atrophic gastritis and in pancreatic metaplasia, respectively. In normal corpus mucosa and in nonatrophic corpus gastritis, PDX-1 was mainly absent. In pancreatic metaplasia, PDX-1 was found in metaplastic cells and in adjacent gastric glands. In contrast to normal gastric corpus mucosa, PDX-1 could be strongly detected in the cytoplasm of the parietal cells surrounding metaplastic areas. Furthermore, PDX-1 expression was found in hyperplastic endocrine cells and in the surrounding gastric glands in chronic atrophic gastritis. Hyperplastic endocrine cells coexpressed the beta-subunit of the gastric H,K-ATPase. We conclude that PDX-1 represents a candidate switch factor for glandular exocrine and endocrine transdifferentiation in chronic gastritis and that an impaired parietal cell differentiation might play a key role in disturbed gastric morphogenic processes.

Collagen IV synthesis is restricted to the enteroendocrine pathway during multilineage differentiation of human colorectal epithelial stem cells.

The human large intestine is lined by a rapidly renewing epithelial monolayer where cell loss is precisely balanced with cell production. The continuous supply of new cells is produced by undifferentiated multipotent stem cells via a coordinated program of proliferation and differentiation yielding three epithelial lineages: absorptive, goblet and enteroendocrine. Cell-matrix interactions have been suggested to be regulators of the multilineage differentiation program of the colorectal crypt but the expression of matrix proteins or their receptors does not appear to have the subtlety expected for this task. We have developed an in vitro model system of intestinal epithelial stem cells to facilitate the direct analysis of stem cells undergoing lineage commitment and differentiation. Using this culture system, we can now directly investigate the role of cell-matrix signalling in stem-cell decisions. In this study, collagen-IV synthesis has been followed in monolayers of multipotent cells that have been induced to differentiate into absorptive, goblet and enteroendocrine cells. Our experiments demonstrate that commitment to the enteroendocrine lineage is specifically accompanied by the expression of type-IV collagen that remains enteroendocrine-cell associated. Undifferentiated cells, absorptive cells and goblet cells do not express collagen IV. To confirm that the differential lineage-specific expression of collagen IV observed in the model system was representative of the in vivo situation, collagen-IV synthesis was analysed in isolated human colorectal crypts and tissue sections using immunocytochemistry and in situ hybridisation. These studies confirmed the in vitro findings, in that implementation of the enteroendocrine differentiation program involves synthesis and accumulation of a collagen-IV matrix. Thus, human colorectal enteroendocrine cells are unique in the colorectal crypt in that they assemble a cell-associated collagen-IV-rich matrix not observed on other colorectal epithelial cells. This study provides the first evidence for differential matrix synthesis between colorectal epithelial lineages in human colorectal epithelium. The specialised pericellular environment of the enteroendocrine cells might explain some of the unique phenotypic characteristics of this cell lineage. Furthermore, these findings suggest a potential mechanism whereby individual epithelial cells could modulate their cell-matrix signalling even while rapidly migrating in heterogeneous sheets over a shared basement membrane.