In JoVE (1)

Other Publications (85)

Articles by Fiona M. Gribble in JoVE

Other articles by Fiona M. Gribble on PubMed

Glucose-sensing in Glucagon-like Peptide-1-secreting Cells

Diabetes. Sep, 2002  |  Pubmed ID: 12196469

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to carbohydrate and fat in the diet. Despite the interest in GLP-1 as an antidiabetic agent, very little is known about the mechanism of stimulus-secretion coupling in L-cells. We investigated the electrophysiological events underlying glucose-induced GLP-1 release in the GLP-1-secreting cell line, GLUTag. Cells were studied using perforated-patch and standard whole-cell patch clamp recordings. GLUTag cells were largely quiescent and hyperpolarized in the absence of glucose. Increasing the glucose concentration between 0 and 20 mmol/l decreased the membrane conductance, caused membrane depolarization, and triggered the generation of action potentials. Action potentials were also triggered by tolbutamide (500 micro mol/l) and were suppressed by diazoxide (340 micro mol/l) or the metabolic inhibitor azide (3 mmol/l), suggesting an involvement of K(ATP) channels. Large tolbutamide-sensitive washout currents developed in standard whole-cell recordings, confirming the presence of K(ATP) channels. RT-PCR detected the K(ATP) channel subunits Kir6.2 and SUR1 and glucokinase. GLP-1 secretion was also stimulated by glucose over the concentration range 0-25 mmol/l and by tolbutamide. Our results suggest that glucose triggers GLP-1 release through closure of K(ATP) channels and action potential generation.

Analysis of the Differential Modulation of Sulphonylurea Block of Beta-cell and Cardiac ATP-sensitive K+ (K(ATP)) Channels by Mg-nucleotides

The Journal of Physiology. Feb, 2003  |  Pubmed ID: 12562963

Sulphonylureas stimulate insulin secretion by binding with high-affinity to the sulphonylurea receptor (SUR) subunit of the ATP-sensitive potassium (K(ATP)) channel and thereby closing the channel pore (formed by four Kir6.2 subunits). In the absence of added nucleotides, the maximal block is around 60-80 %, indicating that sulphonylureas act as partial antagonists. Intracellular MgADP modulated sulphonylurea block, enhancing inhibition of Kir6.2/SUR1 (beta-cell type) and decreasing that of Kir6.2/SUR2A (cardiac-type) channels. We examined the molecular basis of the different response of channels containing SUR1 and SUR2A, by recording currents from inside-out patches excised from Xenopus oocytes heterologously expressing wild-type or chimeric channels. We used the benzamido derivative meglitinide as this drug blocks Kir6.2/SUR1 and Kir6.2/SUR2A currents, reversibly and with similar potency. Our results indicate that transfer of the region containing transmembrane helices (TMs) 8-11 and the following 65 residues of SUR1 into SUR2A largely confers a SUR1-like response to MgADP and meglitinide, whereas the reverse chimera (SUR128) largely endows SUR1 with a SUR2A-type response. This effect was not specific for meglitinide, as tolbutamide was also unable to prevent MgADP activation of Kir6.2/SUR128 currents. The data favour the idea that meglitinide binding to SUR1 impairs either MgADP binding or the transduction pathway between the NBDs and Kir6.2, and that TMs 8-11 are involved in this modulatory response. The results provide a basis for understanding how beta-cell K(ATP) channels show enhanced sulphonylurea inhibition under physiological conditions, whereas cardiac K(ATP) channels exhibit reduced block in intact cells, especially during metabolic inhibition.

Differential Selectivity of Insulin Secretagogues: Mechanisms, Clinical Implications, and Drug Interactions

Journal of Diabetes and Its Complications. Mar-Apr, 2003  |  Pubmed ID: 12623163

The sulphonylurea receptor (SUR) subunits of K(ATP) channels are the targets for several classes of therapeutic drugs. Sulphonylureas close K(ATP) channels in pancreatic beta-cells and are used to stimulate insulin release in type 2 diabetes, whereas the K(ATP) channel opener nicorandil acts as an antianginal agent by opening K(ATP) channels in cardiac and vascular smooth muscle. The predominant type of SUR varies between tissues: SUR1 in beta-cells, SUR2A in cardiac muscle, and SUR2B in smooth muscle. Sulphonylureas and related drugs exhibit differences in tissue specificity, as the drugs interact to varying degrees with different types of SUR. Gliclazide and tolbutamide are beta-cell selective and reversible. Glimepiride, glibenclamide, and repaglinide, however, inhibit cardiac and smooth muscle K(ATP) channels in addition to those in beta-cells and are only slowly reversible. Similar properties have been observed by recording K(ATP) channel activity in intact cells and in Xenopus oocytes expressing cloned K(ATP) channel subunits. While K(ATP) channels in cardiac and smooth muscle are largely closed under physiological conditions (but open during ischaemia), they are activated by antianginal agents such as nicorandil. Under these conditions, they may be inhibited by sulphonylureas that block SUR2-type K(ATP) channels (e.g., glibenclamide). Care should, therefore, be taken when choosing a sulphonylurea if potential interactions with cardiac and smooth muscle K(ATP) channels are to be avoided.

A Novel Glucose-sensing Mechanism Contributing to Glucagon-like Peptide-1 Secretion from the GLUTag Cell Line

Diabetes. May, 2003  |  Pubmed ID: 12716745

Glucagon-like peptide 1 (GLP-1) secretion from intestinal L-cells is triggered by luminal nutrients. We reported previously that glucose-triggered GLP-1 release from the L-cell model GLUTag involves closure of ATP-sensitive K+ (K(ATP)) channels. We show here that GLP-1 secretion and electrical activity of GLUTag cells is triggered not only by metabolizable sugars (glucose or fructose) but also by the nonmetabolizable monosaccharide methyl-alpha-glucopyranoside. Responses to glucose and methyl-alpha-glucopyranoside were impaired by the sodium-glucose cotransporter (SGLT) inhibitor phloridzin. SLGT1 and 3 were detected in GLUTag cells by RT-PCR. Whereas fructose closed K(ATP) channels, methyl-alpha-glucopyranoside increased the membrane conductance and generated an inward current. Low concentrations of glucose and methyl-alpha-glucopyranoside also triggered small inward currents and enhanced the action potential frequency. We conclude that whereas low concentrations of metabolizable sugars trigger GLP-1 secretion via K(ATP) channel closure, SGLT substrates generate small inward currents as a result of the electrogenic action of the transporter. This transporter-associated current can trigger electrical activity and secretion when the concentration of substrate is high or when outward currents are reduced by metabolic closure of the K(ATP) channels. Electrogenic sugar entry via SGLTs provides a novel mechanism for glucose sensing by neuroendocrine cells.

Genetic Cause of Hyperglycaemia and Response to Treatment in Diabetes

Lancet (London, England). Oct, 2003  |  Pubmed ID: 14575972

Type 2 diabetes shows evidence of underlying heterogeneity. No studies have assessed whether different causes for diabetes change the response to oral hypoglycaemic therapy. In a few cases, patients with diabetes caused by mutations in the hepatocyte nuclear factor 1alpha (HNF-1alpha) gene have been described as sensitive to the hypoglycaemic effects of sulphonylureas. We aimed to see whether the glycaemic response to the sulphonylurea gliclazide and the biguanide metformin differed in HNF-1alpha diabetes and type 2 diabetes, and to investigate the mechanism for differences in sulphonylurea sensitivity.

Open to Control--new Hope for Patients with Neonatal Diabetes

The New England Journal of Medicine. Apr, 2004  |  Pubmed ID: 15115828

Relapsing Diabetes Can Result from Moderately Activating Mutations in KCNJ11

Human Molecular Genetics. Apr, 2005  |  Pubmed ID: 15718250

Neonatal diabetes can either remit and hence be transient or else may be permanent. These two phenotypes were considered to be genetically distinct. Abnormalities of 6q24 are the commonest cause of transient neonatal diabetes (TNDM). Mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium channel (K(ATP)), are the commonest cause of permanent neonatal diabetes (PNDM). In addition to diabetes, some KCNJ11 mutations also result in marked developmental delay and epilepsy. These mutations are more severe on functional characterization. We investigated whether mutations in KCNJ11 could also give rise to TNDM. We identified the three novel heterozygous mutations (G53S, G53R, I182V) in three of 11 probands with clinically defined TNDM, who did not have chromosome 6q24 abnormalities. The mutations co-segregated with diabetes within families and were not found in 100 controls. All probands had insulin-treated diabetes diagnosed in the first 4 months and went into remission by 7-14 months. Functional characterization of the TNDM associated mutations was performed by expressing the mutated Kir6.2 with SUR1 in Xenopus laevis oocytes. All three heterozygous mutations resulted in a reduction in the sensitivity to ATP when compared with wild-type (IC(50) approximately 30 versus approximately 7 microM, P-value for is all <0.01); however, this was less profoundly reduced than with the PNDM associated mutations. In conclusion, mutations in KCNJ11 are the first genetic cause for remitting as well as permanent diabetes. This suggests that a fixed ion channel abnormality can result in a fluctuating glycaemic phenotype. The multiple phenotypes associated with activating KCNJ11 mutations may reflect their severity in vitro.

Metabolism: a Higher Power for Insulin

Nature. Apr, 2005  |  Pubmed ID: 15846327

Mucolipin-1 is a Lysosomal Membrane Protein Required for Intracellular Lactosylceramide Traffic

Traffic (Copenhagen, Denmark). Oct, 2006  |  Pubmed ID: 16978393

Mucolipin-1 is a membrane protein encoded by the gene MCOLN1, mutations in which result in the lysosomal storage disorder mucolipidosis type IV (MLIV). Efficient lysosomal targeting of mucolipin-1 requires di-leucine motifs in both the N-terminal and the C-terminal cytosolic tails. We have shown that aberrant lactosylceramide trafficking in MLIV cells may be rescued by wild-type mucolipin-1 expression but not by mucolipin-1 mistargeted to the plasma membrane or by lysosome-localized mucolipin-1 mutated in its predicted ion pore-selectivity region. Our data demonstrate that the correct localization of mucolipin-1 and the integrity of its ion pore are essential for its physiological function in the late endocytic pathway.

Sodium-coupled Glucose Cotransporters Contribute to Hypothalamic Glucose Sensing

Diabetes. Dec, 2006  |  Pubmed ID: 17130483

Specialized neurons within the hypothalamus have the ability to sense and respond to changes in ambient glucose concentrations. We investigated the mechanisms underlying glucose-triggered activity in glucose-excited neurons, using primary cultures of rat hypothalamic neurons monitored by fluorescence calcium imaging. We found that 35% (738 of 2,139) of the neurons were excited by increasing glucose from 3 to 15 mmol/l, but only 9% (6 of 64) of these glucose-excited neurons were activated by tolbutamide, suggesting the involvement of a ATP-sensitive K(+) channel-independent mechanism. alpha-Methylglucopyranoside (alphaMDG; 12 mmol/l), a nonmetabolizable substrate of sodium glucose cotransporters (SGLTs), mimicked the effect of high glucose in 67% of glucose-excited neurons, and both glucose- and alphaMDG-triggered excitation were blocked by Na(+) removal or by the SGLT inhibitor phloridzin (100 nmol/l). In the presence of 0.5 mmol/l glucose and tolbutamide, responses could also be triggered by 3.5 mmol/l alphaMDG, supporting a role for an SGLT-associated mechanism at low as well as high substrate concentrations. Using RT-PCR, we detected SGLT1, SGLT3a, and SGLT3b in both cultured neurons and adult rat hypothalamus. Our findings suggest a novel role for SGLTs in glucose sensing by hypothalamic glucose-excited neurons.

An SCN9A Channelopathy Causes Congenital Inability to Experience Pain

Nature. Dec, 2006  |  Pubmed ID: 17167479

The complete inability to sense pain in an otherwise healthy individual is a very rare phenotype. In three consanguineous families from northern Pakistan, we mapped the condition as an autosomal-recessive trait to chromosome 2q24.3. This region contains the gene SCN9A, encoding the alpha-subunit of the voltage-gated sodium channel, Na(v)1.7, which is strongly expressed in nociceptive neurons. Sequence analysis of SCN9A in affected individuals revealed three distinct homozygous nonsense mutations (S459X, I767X and W897X). We show that these mutations cause loss of function of Na(v)1.7 by co-expression of wild-type or mutant human Na(v)1.7 with sodium channel beta(1) and beta(2) subunits in HEK293 cells. In cells expressing mutant Na(v)1.7, the currents were no greater than background. Our data suggest that SCN9A is an essential and non-redundant requirement for nociception in humans. These findings should stimulate the search for novel analgesics that selectively target this sodium channel subunit.

TCF7L2 Polymorphisms Modulate Proinsulin Levels and Beta-cell Function in a British Europid Population

Diabetes. Jul, 2007  |  Pubmed ID: 17416797

Rapidly accumulating evidence shows that common T-cell transcription factor (TCF)7L2 polymorphisms confer risk of type 2 diabetes through unknown mechanisms. We examined the association between four TCF7L2 single nucleotide polymorphisms (SNPs), including rs7903146, and measures of insulin sensitivity and insulin secretion in 1,697 Europid men and women of the population-based MRC (Medical Research Council)-Ely study. The T-(minor) allele of rs7903146 was strongly and positively associated with fasting proinsulin (P = 4.55 x 10(-9)) and 32,33 split proinsulin (P = 1.72 x 10(-4)) relative to total insulin levels; i.e., differences between T/T and C/C homozygotes amounted to 21.9 and 18.4% respectively. Notably, the insulin-to-glucose ratio (IGR) at 30-min oral glucose tolerance test (OGTT), a frequently used surrogate of first-phase insulin secretion, was not associated with the TCF7L2 SNP (P > 0.7). However, the insulin response (IGR) at 60-min OGTT was significantly lower in T-allele carriers (P = 3.5 x 10(-3)). The T-allele was also associated with higher A1C concentrations (P = 1.2 x 10(-2)) and reduced beta-cell function, assessed by homeostasis model assessment of beta-cell function (P = 2.8 x 10(-2)). Similar results were obtained for the other TCF7L2 SNPs. Of note, both major genes involved in proinsulin processing (PC1, PC2) contain TCF-binding sites in their promoters. Our findings suggest that the TCF7L2 risk allele may predispose to type 2 diabetes by impairing beta-cell proinsulin processing. The risk allele increases proinsulin levels and diminishes the 60-min but not 30-min insulin response during OGTT. The strong association between the TCF7L2 risk allele and fasting proinsulin but not insulin levels is notable, as, in this unselected and largely normoglycemic population, external influences on beta-cell stress are unlikely to be major factors influencing the efficiency of proinsulin processing.

Impairment of the Ubiquitin-proteasome Pathway is a Downstream Endoplasmic Reticulum Stress Response Induced by Extracellular Human Islet Amyloid Polypeptide and Contributes to Pancreatic Beta-cell Apoptosis

Diabetes. Sep, 2007  |  Pubmed ID: 17563070

Human islet amyloid polypeptide (hIAPP) aggregation plays a major role in the development of islet amyloidosis in type 2 diabetes. It is known that extracellular hIAPP oligomers are toxic to pancreatic beta-cells and associated with apoptosis. We therefore investigated the molecular mechanism by which extracellular hIAPP mediates pancreatic beta-cell apoptosis.

No Differences in Mortality Between Users of Pancreatic-specific and Non-pancreatic-specific Sulphonylureas: a Cohort Analysis

Diabetes, Obesity & Metabolism. Apr, 2008  |  Pubmed ID: 18093208

To assess whether users of pancreatic-specific sulphonylureas are at reduced risk of mortality and cardiovascular mortality compared with users of non-specific sulphonylureas, we conducted a cohort study in the population of Tayside, Scotland. We identified 3331 patients with type 2 diabetes who were newly treated with sulphonylureas between 1994 and 2001 and categorized them into those treated with only pancreatic-specific sulphonylureas and those treated with only non-specific sulphonylureas. The risks of mortality and cardiovascular mortality were compared in a survival analysis. There were 2914 patients treated with pancreatic-specific sulphonylureas only, of which 683 (23.4%) died. Of 186 patients treated with non-specific drugs only, 40 (21.5%) died. After adjusting for confounding factors, the adjusted risk ratios (with 95% CI) for mortality and cardiovascular mortality were 0.84 (0.61 to 1.17) and 0.81 (0.59 to 1.11) among the non-specific users compared with the pancreatic-specific users. This provides no evidence that there are differences between the two sulphonylureas types.

Glucose Sensing in L Cells: a Primary Cell Study

Cell Metabolism. Dec, 2008  |  Pubmed ID: 19041768

Glucagon-like peptide-1 (GLP-1) is an enteric hormone that stimulates insulin secretion and improves glycaemia in type 2 diabetes. Although GLP-1-based treatments are clinically available, alternative strategies to increase endogenous GLP-1 release from L cells are hampered by our limited physiological understanding of this cell type. By generating transgenic mice with L cell-specific expression of a fluorescent protein, we studied the characteristics of primary L cells by electrophysiology, fluorescence calcium imaging, and expression analysis and show that single L cells are electrically excitable and glucose responsive. Sensitivity to tolbutamide and low-millimolar concentrations of glucose and alpha-methylglucopyranoside, assessed in single L cells and by hormone secretion from primary cultures, suggested that GLP-1 release is regulated by the activity of sodium glucose cotransporter 1 and ATP-sensitive K(+) channels, consistent with their high expression levels in purified L cells by quantitative RT-PCR. These and other pathways identified using this approach will provide exciting opportunities for future physiological and therapeutic exploration.

Nutritional Regulation of Glucagon-like Peptide-1 Secretion

The Journal of Physiology. Jan, 2009  |  Pubmed ID: 19001044

Glucagon-like peptide-1 (GLP-1), released from L-cells in the intestinal epithelium, plays an important role in postprandial glucose homeostasis and appetite control. Following the recent therapeutic successes of antidiabetic drugs aimed at either mimicking GLP-1 or preventing its degradation, attention is now turning towards the L-cell, and addressing whether it would be both possible and beneficial to stimulate the endogenous release of GLP-1 in vivo. Understanding the mechanisms underlying GLP-1 release from L-cells is key to this type of approach, and the use of cell line models has led to the identification of a variety of pathways that may underlie the physiological responses of L-cells to food ingestion. This review focuses on our current understanding of the signalling mechanisms that underlie L-cell nutrient responsiveness.

Oral Glutamine Increases Circulating Glucagon-like Peptide 1, Glucagon, and Insulin Concentrations in Lean, Obese, and Type 2 Diabetic Subjects

The American Journal of Clinical Nutrition. Jan, 2009  |  Pubmed ID: 19056578

Incretin hormones, such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play an important role in meal-related insulin secretion. We previously demonstrated that glutamine is a potent stimulus of GLP-1 secretion in vitro.

Intolerant of Glucose and Gasping for Oxygen

Nature Medicine. Mar, 2009  |  Pubmed ID: 19265823

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes-associated Variants

Diabetes. Sep, 2009  |  Pubmed ID: 19542200

Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele.

Hypogonadotropic Hypogonadism Due to a Novel Missense Mutation in the First Extracellular Loop of the Neurokinin B Receptor

The Journal of Clinical Endocrinology and Metabolism. Oct, 2009  |  Pubmed ID: 19755480

The neurokinin B (NKB) receptor, encoded by TACR3, is widely expressed within the central nervous system, including hypothalamic nuclei involved in regulating GnRH release. We have recently reported two mutations in transmembrane segments of the receptor and a missense mutation in NKB in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH).

Molecular Mechanisms Underlying Nutrient-stimulated Incretin Secretion

Expert Reviews in Molecular Medicine. Jan, 2010  |  Pubmed ID: 20047700

The incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are released from enteroendocrine cells in the intestinal epithelium in response to nutrient ingestion. The actions of GLP-1 and GIP - not only on local gut physiology but also on glucose homeostasis, appetite control and fat metabolism - have made these hormones an attractive area for drug discovery programmes. The potential range of strategies to target the secretion of these hormones therapeutically has been limited by an incomplete understanding of the mechanisms underlying their release. The use of organ and whole-animal perfusion techniques, cell line models and primary L- and K-cells has led to the identification of a variety of pathways involved in the sensing of carbohydrate, fat and protein in the gut lumen. This review focuses on our current understanding of these signalling mechanisms that might underlie nutrient responsiveness of L- and K-cells.

Alpha2A-adrenergic Receptors and Type 2 Diabetes

The New England Journal of Medicine. Jan, 2010  |  Pubmed ID: 20107223

Pain Perception is Altered by a Nucleotide Polymorphism in SCN9A

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2010  |  Pubmed ID: 20212137

The gene SCN9A is responsible for three human pain disorders. Nonsense mutations cause a complete absence of pain, whereas activating mutations cause severe episodic pain in paroxysmal extreme pain disorder and primary erythermalgia. This led us to investigate whether single nucleotide polymorphisms (SNPs) in SCN9A were associated with differing pain perception in the general population. We first genotyped 27 SCN9A SNPs in 578 individuals with a radiographic diagnosis of osteoarthritis and a pain score assessment. A significant association was found between pain score and SNP rs6746030; the rarer A allele was associated with increased pain scores compared to the commoner G allele (P = 0.016). This SNP was then further genotyped in 195 pain-assessed people with sciatica, 100 amputees with phantom pain, 179 individuals after lumbar discectomy, and 205 individuals with pancreatitis. The combined P value for increased A allele pain was 0.0001 in the five cohorts tested (1277 people in total). The two alleles of the SNP rs6746030 alter the coding sequence of the sodium channel Nav1.7. Each was separately transfected into HEK293 cells and electrophysiologically assessed by patch-clamping. The two alleles showed a difference in the voltage-dependent slow inactivation (P = 0.042) where the A allele would be predicted to increase Nav1.7 activity. Finally, we genotyped 186 healthy females characterized by their responses to a diverse set of noxious stimuli. The A allele of rs6746030 was associated with an altered pain threshold and the effect mediated through C-fiber activation. We conclude that individuals experience differing amounts of pain, per nociceptive stimulus, on the basis of their SCN9A rs6746030 genotype.

Leptin Directly Depolarizes Preproglucagon Neurons in the Nucleus Tractus Solitarius: Electrical Properties of Glucagon-like Peptide 1 Neurons

Diabetes. Aug, 2010  |  Pubmed ID: 20522593

Glucagon-like peptide (GLP)-1 inhibits food intake, acting both in the periphery and within the central nervous system. It is unclear if gut-derived GLP-1 can enter the brain, or whether GLP-1 from preproglucagon (PPG) cells in the lower brainstem is required to activate central GLP-1 receptors. Brainstem PPG neurons, however, have been poorly characterized, due to the difficulties in identifying these cells while viable. This study provides data on the electrical properties of brainstem PPG cells and their regulation by orexigenic and anorexigenic peptides.

Role of Phosphodiesterase and Adenylate Cyclase Isozymes in Murine Colonic Glucagon-like Peptide 1 Secreting Cells

British Journal of Pharmacology. May, 2011  |  Pubmed ID: 21054345

Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine L-cells after food intake. Increasing GLP-1 signalling either through inhibition of the GLP-1 degrading enzyme dipeptidyl-peptidase IV or injection of GLP-1-mimetics has recently been successfully introduced for the treatment of type 2 diabetes. Boosting secretion from the L-cell has so far not been exploited, due to our incomplete understanding of L-cell physiology. Elevation of cyclic adenosine monophosphate (cAMP) has been shown to be a strong stimulus for GLP-1 secretion and here we investigate the activities of adenylate cyclase (AC) and phosphodiesterase (PDE) isozymes likely to shape cAMP responses in L-cells.

Glutamine Triggers and Potentiates Glucagon-like Peptide-1 Secretion by Raising Cytosolic Ca2+ and CAMP

Endocrinology. Feb, 2011  |  Pubmed ID: 21209017

L-glutamine stimulates glucagon-like peptide 1 (GLP-1) secretion in human subjects and cell lines. As recent advances have enabled the study of primary GLP-1-releasing L cells, this study aimed to characterize glutamine-sensing pathways in native murine L cells. L cells were identified using transgenic mice with cell-specific expression of fluorescent markers. Cells were studied in primary colonic cultures from adult mice, or purified by flow cytometry for expression analysis. Intracellular Ca(2+) was monitored in cultures loaded with Fura2, and cAMP was studied using Förster resonance energy transfer sensors expressed in GLUTag cells. Asparagine, phenylalanine, and glutamine (10 mm) triggered GLP-1 release from primary cultures, but glutamine was the most efficacious, increasing secretion 1.9-fold with an EC(50) of 0.19 mm. Several amino acids triggered Ca(2+) changes in L cells, comparable in magnitude to that induced by glutamine. Glutamine-induced Ca(2+) responses were abolished in low Na(+) solution and attenuated in Ca(2+) free solution, suggesting a role for Na(+) dependent uptake and Ca(2+) influx. The greater effectiveness of glutamine as a secretagogue was paralleled by its ability to increase cAMP in GLUTag cells. Glutamine elevated intracellular cAMP to 36% of that produced by a maximal stimulus, whereas asparagine only increased intracellular cAMP by 24% and phenylalanine was without effect. Glutamine elevates both cytosolic Ca(2+) and cAMP in L cells, which may account for the effectiveness of glutamine as a GLP-1 secretagogue. Therapeutic agents like glutamine that target synergistic pathways in L cells might play a future role in the treatment of type 2 diabetes.

Glutamine Reduces Postprandial Glycemia and Augments the Glucagon-like Peptide-1 Response in Type 2 Diabetes Patients

The Journal of Nutrition. Jul, 2011  |  Pubmed ID: 21593352

Impaired glucagon-like peptide (GLP-1) secretion or response may contribute to ineffective insulin release in type 2 diabetes. The conditionally essential amino acid glutamine stimulates GLP-1 secretion in vitro and in vivo. In a randomized, crossover study, we evaluated the effect of oral glutamine, with or without sitagliptin (SIT), on postprandial glycemia and GLP-1 concentration in 15 type 2 diabetes patients (glycated hemoglobin 6.5 ± 0.6%). Participants ingested a low-fat meal (5% fat) after receiving either water (control), 30 g l-glutamine (Gln-30), 15 g L-glutamine (Gln-15), 100 mg SIT, or 100 mg SIT and 15 g L-glutamine (SIT+Gln-15). Studies were conducted 1-2 wk apart. Blood was collected at baseline and postprandially for 180 min for measurement of circulating glucose, insulin, C-peptide, glucagon, and total and active GLP-1. Gln-30 and SIT+Gln-15 reduced the early (t = 0-60 min) postprandial glycemic response compared with control. All Gln treatments enhanced the postprandial insulin response from t = 60-180 min but had no effect on the C-peptide response compared with control. The postprandial glucagon concentration was increased by Gln-30 and Gln-15 compared with control, but the insulin:glucagon ratio was not affected by any treatment. In contrast to Gln-30, which tended to increase the total GLP-1 AUC, SIT tended to decrease the total GLP-1 AUC relative to control (both P = 0.03). Gln-30 and SIT increased the active GLP-1 AUC compared with control (P = 0.008 and P = 0.01, respectively). In summary, Gln-30 decreased the early postprandial glucose response, enhanced late postprandial insulinemia, and augmented postprandial active GLP-1 responses compared with control. These findings suggest that glutamine may be a novel agent for stimulating GLP-1 concentration and limiting postprandial glycemia in type 2 diabetes.

CCK Stimulation of GLP-1 Neurons Involves α1-adrenoceptor-mediated Increase in Glutamatergic Synaptic Inputs

Diabetes. Nov, 2011  |  Pubmed ID: 21885869

Glucagon-like peptide 1 (GLP-1) is involved in the central regulation of food intake. It is produced within the brain by preproglucagon (PPG) neurons, which are located primarily within the brain stem. These neurons project widely throughout the brain, including to the appetite centers in the hypothalamus, and are believed to convey signals related to satiety. Previous work demonstrated that they are directly activated by leptin and electrical activity of the afferent vagus. Another satiety hormone, cholecystokinin (CCK), has also been linked to activation of brain stem neurons, suggesting that it might act partially via centrally projecting neurons from the nucleus tractus solitarius (NTS). The aim of this study was to investigate the neuronal circuitry linking CCK to the population of NTS-PPG neurons.

Interleukin-6 Enhances Insulin Secretion by Increasing Glucagon-like Peptide-1 Secretion from L Cells and Alpha Cells

Nature Medicine. Oct, 2011  |  Pubmed ID: 22037645

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Na(+)-D-glucose Cotransporter SGLT1 is Pivotal for Intestinal Glucose Absorption and Glucose-dependent Incretin Secretion

Diabetes. Jan, 2012  |  Pubmed ID: 22124465

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

Nutrient Detection by Incretin Hormone Secreting Cells

Physiology & Behavior. Jun, 2012  |  Pubmed ID: 22182802

The hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulintropic polypeptide (GIP) are secreted after a meal. Like other enteroendocrine hormones they help to orchestrate the bodies' response to the availability of newly absorbable nutrients and are noteworthy as they stimulate postprandial insulin secretion, underlying what is known as the incretin effect. GLP-1-mimetics are now widely used in the treatment of type 2 diabetes and advantages over older insulinotropic therapies include weight loss. An alternative treatment regime might be the recruitment of endogenous GLP-1, however, very little is known about the physiological control of enteroendocrine responses. This review focuses on the molecular mechanisms to detect nutrient arrival in the gut that have been implicated within the incretin secreting cells.

Short-chain Fatty Acids Stimulate Glucagon-like Peptide-1 Secretion Via the G-protein-coupled Receptor FFAR2

Diabetes. Feb, 2012  |  Pubmed ID: 22190648

Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of "indigestible" prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1-secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.

Intestinal Sensing of Nutrients

Handbook of Experimental Pharmacology. 2012  |  Pubmed ID: 22249821

Ingestion of a meal triggers a range of physiological responses both within and outside the gut, and results in the remote modulation of appetite and glucose homeostasis. Luminal contents are sensed by specialised chemosensitive cells scattered throughout the intestinal epithelium. These enteroendocrine and tuft cells make direct contact with the gut lumen and release a range of chemical mediators, which can either act in a paracrine fashion interacting with neighbouring cells and nerve endings or as classical circulating hormones. At the molecular level, the chemosensory machinery involves multiple and complex signalling pathways including activation of G-protein-coupled receptors and solute carrier transporters. This chapter will discuss our current knowledge of the molecular mechanisms underlying intestinal chemosensation with a particular focus on the relatively well-characterised nutrient-triggered secretion from the enteroendocrine system.

G-protein-coupled Receptors in Intestinal Chemosensation

Cell Metabolism. Apr, 2012  |  Pubmed ID: 22482725

Food intake is detected by the chemical senses of taste and smell and subsequently by chemosensory cells in the gastrointestinal tract that link the composition of ingested foods to feedback circuits controlling gut motility/secretion, appetite, and peripheral nutrient disposal. G-protein-coupled receptors responsive to a range of nutrients and other food components have been identified, and many are localized to intestinal chemosensory cells, eliciting hormonal and neuronal signaling to the brain and periphery. This review examines the role of G-protein-coupled receptors as signaling molecules in the gut, with a particular focus on pathways relevant to appetite and glucose homeostasis.

Overlap of Endocrine Hormone Expression in the Mouse Intestine Revealed by Transcriptional Profiling and Flow Cytometry

Endocrinology. Jul, 2012  |  Pubmed ID: 22685263

The intestine secretes a range of hormones with important local and distant actions, including the control of insulin secretion and appetite. A number of enteroendocrine cell types have been described, each characterized by a distinct hormonal signature, such as K-cells producing glucose-dependent insulinotropic polypeptide (GIP), L-cells producing glucagon-like peptide-1 (GLP-1), and I-cells producing cholecystokinin (CCK). To evaluate similarities between L-, K-, and other enteroendocrine cells, primary murine L- and K-cells, and pancreatic α- and β-cells, were purified and analyzed by flow cytometry and microarray-based transcriptomics. By microarray expression profiling, L cells from the upper small intestinal (SI) more closely resembled upper SI K-cells than colonic L-cells. Upper SI L-cell populations expressed message for hormones classically localized to different enteroendocrine cell types, including GIP, CCK, secretin, and neurotensin. By immunostaining and fluorescence-activated cell sorting analysis, most colonic L-cells contained GLP-1 and PeptideYY In the upper SI, most L-cells contained CCK, approximately 10% were GIP positive, and about 20% were PeptideYY positive. Upper SI K-cells exhibited approximately 10% overlap with GLP-1 and 6% overlap with somatostatin. Enteroendocrine-specific transcription factors were identified from the microarrays, of which very few differed between the enteroendocrine cell populations. Etv1, Prox1, and Pax4 were significantly enriched in L-cells vs. K cells by quantitative RT-PCR. In summary, our data indicate a strong overlap between upper SI L-, K-, and I-cells and suggest they may rather comprise a single cell type, within which individual cells exhibit a hormonal spectrum that may reflect factors such as location along the intestine and exposure to dietary nutrients.

Neurochemical Characterization of Body Weight-regulating Leptin Receptor Neurons in the Nucleus of the Solitary Tract

Endocrinology. Oct, 2012  |  Pubmed ID: 22869346

The action of peripherally released leptin at long-form leptin receptors (LepRb) within the brain represents a fundamental axis in the regulation of energy homeostasis and body weight. Efforts to delineate the neuronal mediators of leptin action have recently focused on extrahypothalamic populations and have revealed that leptin action within the nucleus of the solitary tract (NTS) is critical for normal appetite and body weight regulation. To elucidate the neuronal circuits that mediate leptin action within the NTS, we employed multiple transgenic reporter lines to characterize the neurochemical identity of LepRb-expressing NTS neurons. LepRb expression was not detected in energy balance-associated NTS neurons that express cocaine- and amphetamine-regulated transcript, brain-derived neurotrophic factor, neuropeptide Y, nesfatin, catecholamines, γ-aminobutyric acid, prolactin-releasing peptide, or nitric oxide synthase. The population of LepRb-expressing NTS neurons was comprised of subpopulations marked by a proopiomelanocortin-enhanced green fluorescent protein (EGFP) transgene and distinct populations that express proglucagon and/or cholecystokinin. The significance of leptin action on these three populations of NTS neurons was assessed in leptin-deficient Ob/Ob mice, revealing increased NTS proglucagon and cholecystokinin, but not proopiomelanocortin, expression. These data provide new insight into the appetitive brainstem circuits engaged by leptin.

The Gut Endocrine System As a Coordinator of Postprandial Nutrient Homoeostasis

The Proceedings of the Nutrition Society. Nov, 2012  |  Pubmed ID: 22906726

Hormones from the gastrointestinal (GI) tract are released following food ingestion and trigger a range of physiological responses including the coordination of appetite and glucose homoeostasis. The aim of this review is to discuss the pathways by which food ingestion triggers secretion of cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) and the altered patterns of gut hormone release observed following gastric bypass surgery. Our understanding of how ingested nutrients trigger secretion of these gut hormones has increased dramatically, as a result of physiological studies in human subjects and animal models and in vitro studies on cell lines and primary intestinal cultures. Specialised enteroendocrine cells located within the gut epithelium are capable of directly detecting a range of nutrient stimuli through a range of receptors and transporters. It is concluded that the arrival of nutrients at the apical surface of enteroendocrine cells is a major stimulus for gut hormone release, thereby coupling these endocrine signals to the arrival of absorbed nutrients in the bloodstream.

Molecular Mechanisms of Incretin Hormone Secretion

Current Opinion in Pharmacology. Dec, 2013  |  Pubmed ID: 24035446

Incretin peptides (glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)) are secreted from enteroendocrine cells in the intestinal epithelium, and help to coordinate metabolic responses to food ingestion. A number of molecular mechanisms have recently been defined that underlie carbohydrate, lipid and protein sensing in gut endocrine cells. Knockout mice lacking sodium glucose tranporter-1 (SGLT-1) or the short chain fatty acid sensing receptor FFAR2 (GPR43), for example, have highlighted the importance of these molecules in incretin secretion. This review outlines our current understanding of sensory pathways in incretin secreting cells and highlights the therapeutic potential of targeting them for the development of novel therapies for obesity and diabetes.

Oligopeptides Stimulate Glucagon-like Peptide-1 Secretion in Mice Through Proton-coupled Uptake and the Calcium-sensing Receptor

Diabetologia. Dec, 2013  |  Pubmed ID: 24045836

Ingested protein is a well-recognised stimulus for glucagon-like peptide-1 (GLP-1) release from intestinal L cells. This study aimed to characterise the molecular mechanisms employed by L cells to detect oligopeptides.

A Tag to Track Short Chain Fatty Acid Sensors

Endocrinology. Oct, 2013  |  Pubmed ID: 24058154

Towards the Harnessing of Gut Feelings

Current Opinion in Pharmacology. Dec, 2013  |  Pubmed ID: 24140240

Generation of L Cells in Mouse and Human Small Intestine Organoids

Diabetes. Feb, 2014  |  Pubmed ID: 24130334

Upon a nutrient challenge, L cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L cells from three-dimensional cultures of mouse and human intestinal crypts. We show that short-chain fatty acids selectively increase the number of L cells, resulting in an elevation of GLP-1 release. This is accompanied by the upregulation of transcription factors associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L cells in mouse and human crypts as a potential basis for novel therapeutic strategies in patients with type 2 diabetes.

Identification and Characterization of GLP-1 Receptor-expressing Cells Using a New Transgenic Mouse Model

Diabetes. Apr, 2014  |  Pubmed ID: 24296712

GLP-1 is an intestinal hormone with widespread actions on metabolism. Therapies based on GLP-1 are highly effective because they increase glucose-dependent insulin secretion in people with type 2 diabetes, but many reports suggest that GLP-1 has additional beneficial or, in some cases, potentially dangerous actions on other tissues, including the heart, vasculature, exocrine pancreas, liver, and central nervous system. Identifying which tissues express the GLP-1 receptor (GLP1R) is critical for the development of GLP-1-based therapies. Our objective was to use a method independent of GLP1R antibodies to identify and characterize the targets of GLP-1 in mice. Using newly generated glp1r-Cre mice crossed with fluorescent reporter strains, we show that major sites of glp1r expression include pancreatic β- and δ-cells, vascular smooth muscle, cardiac atrium, gastric antrum/pylorus, enteric neurones, and vagal and dorsal root ganglia. In the central nervous system, glp1r-fluorescent cells were abundant in the area postrema, arcuate nucleus, paraventricular nucleus, and ventromedial hypothalamus. Sporadic glp1r-fluorescent cells were found in pancreatic ducts. No glp1r-fluorescence was observed in ventricular cardiomyocytes. Enteric and vagal neurons positive for glp1r were activated by GLP-1 and may contribute to intestinal and central responses to locally released GLP-1, such as regulation of intestinal secretomotor activity and appetite.

Fructose Stimulates GLP-1 but Not GIP Secretion in Mice, Rats, and Humans

American Journal of Physiology. Gastrointestinal and Liver Physiology. Apr, 2014  |  Pubmed ID: 24525020

Nutrients often stimulate gut hormone secretion, but the effects of fructose are incompletely understood. We studied the effects of fructose on a number of gut hormones with particular focus on glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). In healthy humans, fructose intake caused a rise in blood glucose and plasma insulin and GLP-1, albeit to a lower degree than isocaloric glucose. Cholecystokinin secretion was stimulated similarly by both carbohydrates, but neither peptide YY3-36 nor glucagon secretion was affected by either treatment. Remarkably, while glucose potently stimulated GIP release, fructose was without effect. Similar patterns were found in the mouse and rat, with both fructose and glucose stimulating GLP-1 secretion, whereas only glucose caused GIP secretion. In GLUTag cells, a murine cell line used as model for L cells, fructose was metabolized and stimulated GLP-1 secretion dose-dependently (EC50 = 0.155 mM) by ATP-sensitive potassium channel closure and cell depolarization. Because fructose elicits GLP-1 secretion without simultaneous release of glucagonotropic GIP, the pathways underlying fructose-stimulated GLP-1 release might be useful targets for type 2 diabetes mellitus and obesity drug development.

Insulin-like Peptide 5 is an Orexigenic Gastrointestinal Hormone

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2014  |  Pubmed ID: 25028498

The gut endocrine system is emerging as a central player in the control of appetite and glucose homeostasis, and as a rich source of peptides with therapeutic potential in the field of diabetes and obesity. In this study we have explored the physiology of insulin-like peptide 5 (Insl5), which we identified as a product of colonic enteroendocrine L-cells, better known for their secretion of glucagon-like peptide-1 and peptideYY. i.p. Insl5 increased food intake in wild-type mice but not mice lacking the cognate receptor Rxfp4. Plasma Insl5 levels were elevated by fasting or prolonged calorie restriction, and declined with feeding. We conclude that Insl5 is an orexigenic hormone released from colonic L-cells, which promotes appetite during conditions of energy deprivation.

GLP-1 Receptor Stimulation of the Lateral Parabrachial Nucleus Reduces Food Intake: Neuroanatomical, Electrophysiological, and Behavioral Evidence

Endocrinology. Nov, 2014  |  Pubmed ID: 25116706

The parabrachial nucleus (PBN) is a key nucleus for the regulation of feeding behavior. Inhibitory inputs from the hypothalamus to the PBN play a crucial role in the normal maintenance of feeding behavior, because their loss leads to starvation. Viscerosensory stimuli result in neuronal activation of the PBN. However, the origin and neurochemical identity of the excitatory neuronal input to the PBN remain largely unexplored. Here, we hypothesize that hindbrain glucagon-like peptide 1 (GLP-1) neurons provide excitatory inputs to the PBN, activation of which may lead to a reduction in feeding behavior. Our data, obtained from mice expressing the yellow fluorescent protein in GLP-1-producing neurons, revealed that hindbrain GLP-1-producing neurons project to the lateral PBN (lPBN). Stimulation of lPBN GLP-1 receptors (GLP-1Rs) reduced the intake of chow and palatable food and decreased body weight in rats. It also activated lPBN neurons, reflected by an increase in the number of c-Fos-positive cells in this region. Further support for an excitatory role of GLP-1 in the PBN is provided by electrophysiological studies showing a remarkable increase in firing of lPBN neurons after Exendin-4 application. We show that within the PBN, GLP-1R activation increased gene expression of 2 energy balance regulating peptides, calcitonin gene-related peptide (CGRP) and IL-6. Moreover, nearly 70% of the lPBN GLP-1 fibers innervated lPBN CGRP neurons. Direct intra-lPBN CGRP application resulted in anorexia. Collectively, our molecular, anatomical, electrophysiological, pharmacological, and behavioral data provide evidence for a functional role of the GLP-1R for feeding control in the PBN.

Diabetes Recovery by Age-dependent Conversion of Pancreatic δ-cells into Insulin Producers

Nature. Oct, 2014  |  Pubmed ID: 25141178

Total or near-total loss of insulin-producing β-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which β-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-β-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on β-cell reconstitution from heterologous islet cells after near-total β-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after β-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although β-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-β-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.

Reversible Changes in Pancreatic Islet Structure and Function Produced by Elevated Blood Glucose

Nature Communications. Aug, 2014  |  Pubmed ID: 25145789

Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that β-cell-specific expression of a human activating KATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some β-cells begin expressing glucagon, whilst retaining many β-cell characteristics. Hyperglycaemia, rather than KATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized.

Na+ Current Properties in Islet α- and β-cells Reflect Cell-specific Scn3a and Scn9a Expression

The Journal of Physiology. Nov, 2014  |  Pubmed ID: 25172946

Mouse pancreatic β- and α-cells are equipped with voltage-gated Na(+) currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at -100 mV and -50 mV in β- and α-cells, respectively). Single-cell PCR analyses show that both α- and β-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: β-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na(+) current by 80%. In β-cells, knockout of Scn9a lowers the Na(+) current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a(-/-) islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na(+) channel α subunit in both α- and β-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in β-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.

The Peutz-Jeghers Kinase LKB1 Suppresses Polyp Growth from Intestinal Cells of a Proglucagon-expressing Lineage in Mice

Disease Models & Mechanisms. Nov, 2014  |  Pubmed ID: 25190708

Liver kinase B1 (LKB1; also known as STK11) is a serine/threonine kinase and tumour suppressor that is mutated in Peutz-Jeghers syndrome (PJS), a premalignant syndrome associated with the development of gastrointestinal polyps. Proglucagon-expressing enteroendocrine cells are involved in the control of glucose homeostasis and the regulation of appetite through the secretion of gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY). To determine the role of LKB1 in these cells, we bred mice bearing floxed alleles of Lkb1 against animals carrying Cre recombinase under proglucagon promoter control. These mice (GluLKB1KO) were viable and displayed near-normal growth rates and glucose homeostasis. However, they developed large polyps at the gastro-duodenal junction, and displayed premature mortality (death from 120 days of age). Histological analysis of the polyps demonstrated that they had a PJS-like appearance with an arborising smooth-muscle core. Circulating GLP-1 levels were normal in GluLKB1KO mice and the polyps expressed low levels of the peptide, similar to levels in the neighbouring duodenum. Lineage tracing using a Rosa26tdRFP transgene revealed, unexpectedly, that enterocytes within the polyps were derived from non-proglucagon-expressing precursors, whereas connective tissue was largely derived from proglucagon-expressing precursors. Developmental studies in wild-type mice suggested that a subpopulation of proglucagon-expressing cells undergo epithelial-mesenchymal transition (EMT) to become smooth-muscle-like cells. Thus, it is likely that polyps in the GluLKB1KO mice developed from a unique population of smooth-muscle-like cells derived from a proglucagon-expressing precursor. The loss of LKB1 within this subpopulation seems to be sufficient to drive tumorigenesis.

The Role of Gut Endocrine Cells in Control of Metabolism and Appetite

Experimental Physiology. Sep, 2014  |  Pubmed ID: 25210110

After food is ingested, nutrients pass through the gastrointestinal tract, stimulating the release of a range of peptide hormones. Among their many local, central and peripheral actions, these hormones act to mediate glucose metabolism and satiety. Indeed, it is the modification of gut hormone secretion that is considered partly responsible for the normalization of glycaemic control and the reduction in appetite seen in many patients after certain forms of bariatric surgery. This review describes recent developments in our understanding of the secretion and action of anorexigenic gut hormones, primarily concentrating on glucagon-like peptide-1 (GLP-1).

Glycemic Effects and Safety of L-Glutamine Supplementation with or Without Sitagliptin in Type 2 Diabetes Patients-a Randomized Study

PloS One. 2014  |  Pubmed ID: 25412338

L-glutamine is an efficacious glucagon-like peptide (GLP)-1 secretagogue in vitro. When administered with a meal, glutamine increases GLP-1 and insulin excursions and reduces postprandial glycaemia in type 2 diabetes patients. The aim of the study was to assess the efficacy and safety of daily glutamine supplementation with or without the dipeptidyl peptidase (DPP)-4 inhibitor sitagliptin in well-controlled type 2 diabetes patients.

Bacterial Metabolite Indole Modulates Incretin Secretion from Intestinal Enteroendocrine L Cells

Cell Reports. Nov, 2014  |  Pubmed ID: 25456122

It has long been speculated that metabolites, produced by gut microbiota, influence host metabolism in health and diseases. Here, we reveal that indole, a metabolite produced from the dissimilation of tryptophan, is able to modulate the secretion of glucagon-like peptide-1 (GLP-1) from immortalized and primary mouse colonic L cells. Indole increased GLP-1 release during short exposures, but it reduced secretion over longer periods. These effects were attributed to the ability of indole to affect two key molecular mechanisms in L cells. On the one hand, indole inhibited voltage-gated K(+) channels, increased the temporal width of action potentials fired by L cells, and led to enhanced Ca(2+) entry, thereby acutely stimulating GLP-1 secretion. On the other hand, indole slowed ATP production by blocking NADH dehydrogenase, thus leading to a prolonged reduction of GLP-1 secretion. Our results identify indole as a signaling molecule by which gut microbiota communicate with L cells and influence host metabolism.

Stimulation of GLP-1 Secretion Downstream of the Ligand-gated Ion Channel TRPA1

Diabetes. Apr, 2015  |  Pubmed ID: 25325736

Stimulus-coupled incretin secretion from enteroendocrine cells plays a fundamental role in glucose homeostasis and could be targeted for the treatment of type 2 diabetes. Here, we investigated the expression and function of transient receptor potential (TRP) ion channels in enteroendocrine L cells producing GLP-1. By microarray and quantitative PCR analysis, we identified trpa1 as an L cell-enriched transcript in the small intestine. Calcium imaging of primary L cells and the model cell line GLUTag revealed responses triggered by the TRPA1 agonists allyl-isothiocyanate (mustard oil), carvacrol, and polyunsaturated fatty acids, which were blocked by TRPA1 antagonists. Electrophysiology in GLUTag cells showed that carvacrol induced a current with characteristics typical of TRPA1 and triggered the firing of action potentials. TRPA1 activation caused an increase in GLP-1 secretion from primary murine intestinal cultures and GLUTag cells, an effect that was abolished in cultures from trpa1(-/-) mice or by pharmacological TRPA1 inhibition. These findings present TRPA1 as a novel sensory mechanism in enteroendocrine L cells, coupled to the facilitation of GLP-1 release, which may be exploitable as a target for treating diabetes.

Targeting Development of Incretin-producing Cells Increases Insulin Secretion

The Journal of Clinical Investigation. Jan, 2015  |  Pubmed ID: 25500886

Glucagon-like peptide-1-based (GLP-1-based) therapies improve glycemic control in patients with type 2 diabetes. While these agents augment insulin secretion, they do not mimic the physiological meal-related rise and fall of GLP-1 concentrations. Here, we tested the hypothesis that increasing the number of intestinal L cells, which produce GLP-1, is an alternative strategy to augment insulin responses and improve glucose tolerance. Blocking the NOTCH signaling pathway with the γ-secretase inhibitor dibenzazepine increased the number of L cells in intestinal organoid-based mouse and human culture systems and augmented glucose-stimulated GLP-1 secretion. In a high-fat diet-fed mouse model of impaired glucose tolerance and type 2 diabetes, dibenzazepine administration increased L cell numbers in the intestine, improved the early insulin response to glucose, and restored glucose tolerance. Dibenzazepine also increased K cell numbers, resulting in increased gastric inhibitory polypeptide (GIP) secretion. Using a GLP-1 receptor antagonist, we determined that the insulinotropic effect of dibenzazepine was mediated through an increase in GLP-1 signaling. Together, our data indicate that modulation of the development of incretin-producing cells in the intestine has potential as a therapeutic strategy to improve glycemic control.

An Absorbing Sense of Sweetness

Diabetes. Feb, 2015  |  Pubmed ID: 25614668

Gut Chemosensing Mechanisms

The Journal of Clinical Investigation. Mar, 2015  |  Pubmed ID: 25664852

The enteroendocrine system is the primary sensor of ingested nutrients and is responsible for secreting an array of gut hormones, which modulate multiple physiological responses including gastrointestinal motility and secretion, glucose homeostasis, and appetite. This Review provides an up-to-date synopsis of the molecular mechanisms underlying enteroendocrine nutrient sensing and highlights our current understanding of the neuro-hormonal regulation of gut hormone secretion, including the interaction between the enteroendocrine system and the enteric nervous system. It is hoped that a deeper understanding of how these systems collectively regulate postprandial physiology will further facilitate the development of novel therapeutic strategies.

Activation of the GLP-1 Receptors in the Nucleus of the Solitary Tract Reduces Food Reward Behavior and Targets the Mesolimbic System

PloS One. 2015  |  Pubmed ID: 25793511

The gut/brain peptide, glucagon like peptide 1 (GLP-1), suppresses food intake by acting on receptors located in key energy balance regulating CNS areas, the hypothalamus or the hindbrain. Moreover, GLP-1 can reduce reward derived from food and motivation to obtain food by acting on its mesolimbic receptors. Together these data suggest a neuroanatomical segregation between homeostatic and reward effects of GLP-1. Here we aim to challenge this view and hypothesize that GLP-1 can regulate food reward behavior by acting directly on the hindbrain, the nucleus of the solitary tract (NTS), GLP-1 receptors (GLP-1R). Using two models of food reward, sucrose progressive ratio operant conditioning and conditioned place preference for food in rats, we show that intra-NTS microinjections of GLP-1 or Exendin-4, a stable analogue of GLP-1, inhibit food reward behavior. When the rats were given a choice between palatable food and chow, intra-NTS Exendin-4 treatment preferentially reduced intake of palatable food but not chow. However, chow intake and body weight were reduced by the NTS GLP-1R activation if chow was offered alone. The NTS GLP-1 activation did not alter general locomotor activity and did not induce nausea, measured by PICA. We further show that GLP-1 fibers are in close apposition to the NTS noradrenergic neurons, which were previously shown to provide a monosynaptic connection between the NTS and the mesolimbic system. Central GLP-1R activation also increased NTS expression of dopamine-β-hydroxylase, a key enzyme in noradrenaline synthesis, indicating a biological link between these two systems. Moreover, NTS GLP-1R activation altered the expression of dopamine-related genes in the ventral tegmental area. These data reveal a food reward-suppressing role of the NTS GLP-1R and indicate that the neurobiological targets underlying food reward control are not limited to the mesolimbic system, instead they are distributed throughout the CNS.

LKB1 and AMPKα1 Are Required in Pancreatic Alpha Cells for the Normal Regulation of Glucagon Secretion and Responses to Hypoglycemia

Molecular Metabolism. Apr, 2015  |  Pubmed ID: 25830091

Glucagon release from pancreatic alpha cells is required for normal glucose homoeostasis and is dysregulated in both Type 1 and Type 2 diabetes. The tumour suppressor LKB1 (STK11) and the downstream kinase AMP-activated protein kinase (AMPK), modulate cellular metabolism and growth, and AMPK is an important target of the anti-hyperglycaemic agent metformin. While LKB1 and AMPK have emerged recently as regulators of beta cell mass and insulin secretion, the role of these enzymes in the control of glucagon production in vivo is unclear.

Submembrane ATP and Ca2+ Kinetics in α-cells: Unexpected Signaling for Glucagon Secretion

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Aug, 2015  |  Pubmed ID: 25911612

Cytoplasmic ATP and Ca(2+) are implicated in current models of glucose's control of glucagon and insulin secretion from pancreatic α- and β-cells, respectively, but little is known about ATP and its relation to Ca(2+) in α-cells. We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca(2+) indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca(2+) and ATP ([Ca(2+)]pm; [ATP]pm) in superficial α- and β-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP's controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucose-induced [ATP]pm generation was left shifted in α-cells compared to β-cells. Both cell types showed [Ca(2+)]pm and [ATP]pm oscillations in opposite phase, probably reflecting energy-consuming Ca(2+) transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca(2+)]pm synchronized in the same phase between α- and β-cells. This paradox can be explained by the overriding of Ca(2+) stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca(2+). The data indicate that an α-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia.

Novel SCN9A Mutations Underlying Extreme Pain Phenotypes: Unexpected Electrophysiological and Clinical Phenotype Correlations

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. May, 2015  |  Pubmed ID: 25995458

The importance of NaV1.7 (encoded by SCN9A) in the regulation of pain sensing is exemplified by the heterogeneity of clinical phenotypes associated with its mutation. Gain-of-function mutations are typically pain-causing and have been associated with inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). IEM is usually caused by enhanced NaV1.7 channel activation, whereas mutations that alter steady-state fast inactivation often lead to PEPD. In contrast, nonfunctional mutations in SCN9A are known to underlie congenital insensitivity to pain (CIP). Although well documented, the correlation between SCN9A genotypes and clinical phenotypes is still unclear. Here we report three families with novel SCN9A mutations. In a multiaffected dominant family with IEM, we found the heterozygous change L245 V. Electrophysiological characterization showed that this mutation did not affect channel activation but instead resulted in incomplete fast inactivation and a small hyperpolarizing shift in steady-state slow inactivation, characteristics more commonly associated with PEPD. In two compound heterozygous CIP patients, we found mutations that still retained functionality of the channels, with two C-terminal mutations (W1775R and L1831X) exhibiting a depolarizing shift in channel activation. Two mutations (A1236E and L1831X) resulted in a hyperpolarizing shift in steady-state fast inactivation. To our knowledge, these are the first descriptions of mutations with some retained channel function causing CIP. This study emphasizes the complex genotype-phenotype correlations that exist for SCN9A and highlights the C-terminal cytoplasmic region of NaV1.7 as a critical region for channel function, potentially facilitating analgesic drug development studies.

Effect of Reducing Portion Size at a Compulsory Meal on Later Energy Intake, Gut Hormones, and Appetite in Overweight Adults

Obesity (Silver Spring, Md.). Jul, 2015  |  Pubmed ID: 26054049

Larger portion sizes (PS) are associated with greater energy intake (EI), but little evidence exists on the appetitive effects of PS reduction. This study investigated the impact of reducing breakfast PS on subsequent EI, postprandial gastrointestinal hormone responses, and appetite ratings.

Farnesoid X Receptor Inhibits Glucagon-like Peptide-1 Production by Enteroendocrine L Cells

Nature Communications. Jul, 2015  |  Pubmed ID: 26134028

Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates β-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes.

Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors

Endocrinology. Nov, 2015  |  Pubmed ID: 26280129

Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca(2+). In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca(2+) response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber-mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms.

Can Encapsulated Glutamine Increase GLP-1 Secretion, Improve Glucose Tolerance, and Reduce Meal Size in Healthy Volunteers? A Randomised, Placebo-controlled, Cross-over Trial

Lancet (London, England). Feb, 2015  |  Pubmed ID: 26312890

Obesity is a global concern and can be effectively treated with bariatric surgery, which is expensive and invasive. Weight loss after surgery has been attributed to increased nutrient delivery to the lower small intestine with release of satiety-promoting gut hormones such as glucagon-like peptide 1 (GLP-1). We aimed to assess whether glutamine, a potent secretagogue of GLP-1 in vivo, increases GLP-1 release, improves glucose tolerance, or reduces meal size in volunteers.

Limited Impact on Glucose Homeostasis of Leptin Receptor Deletion from Insulin- or Proglucagon-expressing Cells

Molecular Metabolism. Sep, 2015  |  Pubmed ID: 26413468

The adipose tissue-derived hormone leptin plays an important role in the maintenance of body weight and glucose homeostasis. Leptin mediates its effects by interaction with leptin receptors (LepRb), which are highly expressed in the hypothalamus and other brain centres, and at lower levels in the periphery. Previous studies have used relatively promiscuous or inefficient Cre deleter strains, respectively, to explore the roles of LepR in pancreatic β and α cells. Here, we use two newly-developed Cre lines to explore the role of leptin signalling in insulin and proglucagon-expressing cells.

Distribution and Characterisation of Glucagon-like Peptide-1 Receptor Expressing Cells in the Mouse Brain

Molecular Metabolism. Oct, 2015  |  Pubmed ID: 26500843

Although Glucagon-like peptide 1 is a key regulator of energy metabolism and food intake, the precise location of GLP-1 receptors and the physiological relevance of certain populations is debatable. This study investigated the novel GLP-1R-Cre mouse as a functional tool to address this question.

Lipid Derivatives Activate GPR119 and Trigger GLP-1 Secretion in Primary Murine L-cells

Peptides. Mar, 2016  |  Pubmed ID: 26144594

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid.

High Fat Diet Impairs the Function of Glucagon-like Peptide-1 Producing L-cells

Peptides. Mar, 2016  |  Pubmed ID: 26145551

Glucagon-like peptide-1 (GLP-1) acts as a satiety signal and enhances insulin release. This study examined how GLP-1 production from intestinal L-cells is modified by dietary changes.

Signalling Pathways Involved in the Detection of Peptones by Murine Small Intestinal Enteroendocrine L-cells

Peptides. Mar, 2016  |  Pubmed ID: 26215048

Glucagon like peptide-1 is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. Here, we investigated mechanisms underlying the sensing of peptones by primary small intestinal L-cells. Meat, casein and vegetable-derived peptones (5 mg/ml), the L-amino acids Phe, Trp, Gln and Ala (20 mM each), and the dipeptide glycine-sarcosine (20 mM) stimulated GLP-1 secretion from primary cultures prepared from the small intestine. Further mechanistic studies were performed with meat peptone, and revealed the elevation of intracellular calcium in L-cells. Inhibition of the calcium sensing receptor (CaSR), transient receptor potential (TRP) channels and Q-type voltage gated calcium channels (VGCC) significantly attenuated peptone-stimulated GLP-1 release and reduced intracellular Ca(2+) responses. CaSR inhibition also attenuated the GLP-1 secretory response to Gln. Targeting these pathways in L-cells could be used to increase endogenous production of GLP-1 and offer exploitable avenues for the development of therapeutics to treat diabetes and obesity.

The Effect of Bariatric Surgery on Gastrointestinal and Pancreatic Peptide Hormones

Peptides. Mar, 2016  |  Pubmed ID: 26344355

Bariatric surgery for obesity has proved to be an extremely effective method of promoting long-term weight reduction with additional beneficial metabolic effects, such as improved glucose tolerance and remission of type 2 diabetes. A range of bariatric procedures are in common use, including gastric banding, sleeve gastrectomy and the Roux-en-Y gastric bypass. Although the mechanisms underlying the efficacy of bariatric surgery are unclear, gastrointestinal and pancreatic peptides are thought to play an important role. The aim of this review is to summarise the effects of different bariatric surgery procedures upon gastrointestinal and pancreatic peptides, including ghrelin, gastrin, cholecystokinin (CCK), glucose-dependent insulinotropic hormone (GIP), glucagon-like peptide 1 (GLP-1), peptide YY (PYY), oxyntomodulin, insulin, glucagon and somatostatin.

Enteroendocrine Cells: Chemosensors in the Intestinal Epithelium

Annual Review of Physiology. 2016  |  Pubmed ID: 26442437

The enteroendocrine system orchestrates how the body responds to the ingestion of foods, employing a diversity of hormones to fine-tune a wide range of physiological responses both within and outside the gut. Recent interest in gut hormones has surged with the realization that they modulate glucose tolerance and food intake through a variety of mechanisms, and such hormones are therefore excellent therapeutic candidates for the treatment of diabetes and obesity. Characterizing the roles and functions of different enteroendocrine cells is an essential step in understanding the physiology, pathophysiology, and therapeutics of the gut-brain-pancreas axis.

GLP1- and GIP-producing Cells Rarely Overlap and Differ by Bombesin Receptor-2 Expression and Responsiveness

The Journal of Endocrinology. Jan, 2016  |  Pubmed ID: 26483393

The incretin hormones glucagon-like peptide-1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted from intestinal endocrine cells, the so-called L- and K-cells. The cells are derived from a common precursor and are highly related, and co-expression of the two hormones in so-called L/K-cells has been reported. To investigate the relationship between the GLP1- and GIP-producing cells more closely, we generated a transgenic mouse model expressing a fluorescent marker in GIP-positive cells. In combination with a mouse strain with fluorescent GLP1 cells, we were able to estimate the overlap between the two cell types. Furthermore, we used primary cultured intestinal cells and isolated perfused mouse intestine to measure the secretion of GIP and GLP1 in response to different stimuli. Overlapping GLP1 and GIP cells were rare (∼5%). KCl, glucose and forskolin+IBMX increased the secretion of both GLP1 and GIP, whereas bombesin/neuromedin C only stimulated GLP1 secretion. Expression analysis showed high expression of the bombesin 2 receptor in GLP1 positive cells, but no expression in GIP-positive cells. These data indicate both expressional and functional differences between the GLP1-producing 'L-cell' and the GIP-producing 'K-cell'.

Galanin Inhibits GLP-1 and GIP Secretion Via the GAL1 Receptor in Enteroendocrine L and K Cells

British Journal of Pharmacology. Mar, 2016  |  Pubmed ID: 26661062

Galanin is a widely expressed neuropeptide, which in the gut is thought to modulate gastrointestinal motility and secretion. We aimed to elucidate the poorly characterised mechanisms underlying the inhibitory effect of galanin and the potential involvement of G-protein coupled inwardly rectifying potassium, Kir 3, (GIRK) channels in glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) secretion.

G Protein-coupled Receptors As New Therapeutic Targets for Type 2 Diabetes

Diabetologia. Feb, 2016  |  Pubmed ID: 26661410

G protein-coupled receptors (GPCRs) in the gut-brain-pancreatic axis are key players in the postprandial control of metabolism and food intake. A number of intestinally located receptors have been implicated in the chemo-detection of ingested nutrients, and in the pancreatic islets and nervous system GPCRs play essential roles in the detection of many hormones and neurotransmitters. Because of the diversity, cell-specific expression and 'druggability' of the GPCR superfamily, these receptors are popular targets for therapeutic development. This review will outline current and potential future approaches to develop GPCR agonists for the treatment of type 2 diabetes. This review summarises a presentation given at the 'Novel approaches to treating type 2 diabetes' symposium at the 2015 annual meeting of the EASD. It is accompanied by a commentary by the Session Chair, Michael Nauck (DOI: 10.1007/s00125-015-3823-1 ).

Stimulation of Incretin Secreting Cells

Therapeutic Advances in Endocrinology and Metabolism. Feb, 2016  |  Pubmed ID: 26885360

The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon like peptide-1 (GLP-1) are secreted from enteroendocrine cells in the gut and regulate physiological and homeostatic functions related to glucose control, metabolism and food intake. This review provides a systematic summary of the molecular mechanisms underlying secretion from incretin cells, and an understanding of how they sense and interact with lumen and vascular factors and the enteric nervous system through transporters and G-protein coupled receptors (GPCRs) present on their surface to ultimately culminate in hormone release. Some of the molecules described below such as sodium coupled glucose transporter 1 (SGLT1), G-protein coupled receptor (GPR) 119 and GPR40 are targets of novel therapeutics designed to enhance endogenous gut hormone release. Synthetic ligands at these receptors aimed at treating obesity and type 2 diabetes are currently under investigation.

Proglucagon Promoter Cre-Mediated AMPK Deletion in Mice Increases Circulating GLP-1 Levels and Oral Glucose Tolerance

PloS One. 2016  |  Pubmed ID: 27010458

Enteroendocrine L-cells synthesise and release the gut hormone glucagon-like peptide-1 (GLP-1) in response to food transit. Deletion of the tumour suppressor kinase LKB1 from proglucagon-expressing cells leads to the generation of intestinal polyps but no change in circulating GLP-1 levels. Here, we explore the role of the downstream kinase AMP-activated protein kinase (AMPK) in these cells.

Preproglucagon Neurons in the Hindbrain Have IL-6 Receptor-α and Show Ca2+ Influx in Response to IL-6

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. Jul, 2016  |  Pubmed ID: 27097661

Neuronal circuits in the hypothalamus and hindbrain are of importance for control of food intake, energy expenditure, and fat mass. We have recently shown that treatment with exendin-4 (Ex-4), an analog of the proglucagon-derived molecule glucagon-like peptide 1 (GLP-1), markedly increases mRNA expression of the cytokine interleukin-6 (IL-6) in the hypothalamus and hindbrain and that this increase partly mediates the suppression of food intake and body weight by Ex-4. Endogenous GLP-1 in the central nervous system (CNS) is produced by preproglucagon (PPG) neurons of the nucleus of the solitary tract (NTS) in the hindbrain. These neurons project to various parts of the brain, including the hypothalamus. Outside the brain, IL-6 stimulates GLP-1 secretion from the gut and pancreas. In this study, we aim to investigate whether IL-6 can affect GLP-1-producing PPG neurons in the nucleus of the solitary tract (NTS) in mouse hindbrain via the ligand binding part of the IL-6 receptor, IL-6 receptor-α (IL-6Rα). Using immunohistochemistry, we found that IL-6Rα was localized on PPG neurons of the NTS. Recordings of these neurons in GCaMP3/GLP-1 reporter mice showed that IL-6 enhances cytosolic Ca(2+) concentration in neurons capable of expressing PPG. We also show that the Ca(2+) increase originates from the extracellular space. Furthermore, we found that IL-6Rα was localized on cells in the caudal hindbrain expressing immunoreactive NeuN (a neuronal marker) or CNP:ase (an oligodendrocyte marker). In summary, IL-6Rα is present on PPG neurons in the NTS, and IL-6 can stimulate these cells by increasing influx of Ca(2+) to the cytosol from the extracellular space.

Mechanisms Underlying Glucose-dependent Insulinotropic Polypeptide and Glucagon-like Peptide-1 Secretion

Journal of Diabetes Investigation. Apr, 2016  |  Pubmed ID: 27186350

The incretin hormones, glucose-dependent insulinotropic peptide and glucagon-like peptide-1, are secreted from intestinal K- and L cells, respectively, with the former being most abundant in the proximal small intestine, whereas the latter increase in number towards the distal gut. Although an overlap between K- and L cells can be observed immunohistochemically or in murine models expressing fluorescent markers under the control of the two hormone promoters, the majority (>80%) of labeled cells seems to produce only one of these hormones. Transcriptomic analysis showed a close relationship between small intestinal K- and L cells, and glucose sensing mechanisms appear similar in both cell types with a predominant role of electrogenic glucose uptake through sodium-coupled glucose transporter 1. Similarly, both cell types produce the long-chain fatty acid sensing G-protein-coupled receptors, FFAR1 (GPR40) and FFAR4 (GPR120), but differ in the expression/functionality of other lipid sensing receptors. GPR119 and FFAR2/3, for example, have clearly documented roles in glucagon-like peptide-1 secretion, whereas agonists for the endocannabinoid receptor type 1 have been found to show largely selective inhibition of glucose-dependent insulinotropic peptide secretion. In conclusion, although K- and L cell populations overlap and share key molecular nutrient-sensing mechanisms, subtle differences between the responsiveness of the different cell types might be exploited to differentially modulate glucose-dependent insulinotropic peptide or glucagon-like peptide-1 secretion.

Transcriptomic Profiling of Pancreatic Alpha, Beta and Delta Cell Populations Identifies Delta Cells As a Principal Target for Ghrelin in Mouse Islets

Diabetologia. Oct, 2016  |  Pubmed ID: 27390011

Intra-islet and gut-islet crosstalk are critical in orchestrating basal and postprandial metabolism. The aim of this study was to identify regulatory proteins and receptors underlying somatostatin secretion though the use of transcriptomic comparison of purified murine alpha, beta and delta cells.

GPR119, a Major Enteroendocrine Sensor of Dietary Triglyceride Metabolites Coacting in Synergy With FFA1 (GPR40)

Endocrinology. Dec, 2016  |  Pubmed ID: 27779915

Triglycerides (TGs) are among the most efficacious stimulators of incretin secretion; however, the relative importance of FFA1 (G Protein-coupled Receptor [GPR] 40), FFA4 (GPR120), and GPR119, which all recognize TG metabolites, ie, long-chain fatty acid and 2-monoacylglycerol, respectively, is still unclear. Here, we find all 3 receptors to be highly expressed and highly enriched in fluorescence-activated cell sorting-purified GLP-1 and GIP cells isolated from transgenic reporter mice. In vivo, the TG-induced increase in plasma GIP was significantly reduced in FFA1-deficient mice (to 34%, mean of 4 experiments each with 8-10 animals), in GPR119-deficient mice (to 24%) and in FFA1/FFA4 double deficient mice (to 15%) but not in FFA4-deficient mice. The TG-induced increase in plasma GLP-1 was only significantly reduced in the GPR119-deficient and the FFA1/FFA4 double deficient mice, but not in the FFA1, and FFA4-deficient mice. In mouse colonic crypt cultures the synthetic FFA1 agonists, TAK-875 stimulated GLP-1 secretion to a similar extent as the prototype GLP-1 secretagogue neuromedin C; this, however, only corresponded to approximately half the maximal efficiency of the GPR119 agonist AR231453, whereas the GPR120 agonist Metabolex-209 had no effect. Importantly, when the FFA1 agonist was administered on top of appropriately low doses of the GPR119 agonist, a clear synergistic, ie, more than additive, effect was observed. It is concluded that the 2-monoacylglycerol receptor GPR119 is at least as important as the long-chain fatty acid receptor FFA1 in mediating the TG-induced secretion of incretins and that the 2 receptors act in synergy, whereas FFA4 plays a minor if any role.

Gut Hormone Regulation and Secretion Via FFA1 and FFA4

Handbook of Experimental Pharmacology. 2017  |  Pubmed ID: 27873089

The digestion, absorption and utilisation of dietary triglycerides are controlled by gut hormones, released from enteroendocrine cells along the length of the gastrointestinal tract. Major players in the detection of ingested lipids are the free fatty acid receptors FFA1 and FFA4, which are highly expressed on enteroendocrine cells. These receptors are activated when free fatty acids (FFA) are absorbed across the intestinal epithelium, and provide a dynamic hormonal signal indicating that lipids are arriving in the bloodstream from the gut. This review addresses our current knowledge of how ingested triglycerides modulate gut hormone release via FFA1 and FFA4.

Scaling It Down: New in Vitro Tools to Get the Balance Right

The Biochemical Journal. Jan, 2017  |  Pubmed ID: 28008090

Treatments for diabetes and obesity based on enteroendocrine hormones are a focus of research interest, partly due to the successes of glucagon-like peptide-1 (GLP-1) mimetic peptides in the treatment of diabetes and the correlation of altered enteroendocrine profiles with the positive metabolic outcomes of gastric bypass surgery. It is thought that simultaneous stimulation of more than one receptor might mimic the superior efficacy of the latter and dual or triple-agonist peptides are under investigation. An important step in developing multiple agonists is to establish the relative pharmacological potency and efficacy of new molecules at its different target receptors, and to optimise the balance of activities to achieve the desired treatment outcome. In a recent issue of the Biochemical Journal, Naylor et al. described how they used CRISPR technology to modulate endogenous receptor density in insulinoma cells to get the balance right for a dual incretin peptide engaging both GLP-1- and glucose-dependent insulinotropic polypeptide-receptors.

Glucagon-Like Peptide 1 and Its Analogs Act in the Dorsal Raphe and Modulate Central Serotonin to Reduce Appetite and Body Weight

Diabetes. Apr, 2017  |  Pubmed ID: 28057699

Glucagon-like peptide 1 (GLP-1) and serotonin play critical roles in energy balance regulation. Both systems are exploited clinically as antiobesity strategies. Surprisingly, whether they interact in order to regulate energy balance is poorly understood. Here we investigated mechanisms by which GLP-1 and serotonin interact at the level of the central nervous system. Serotonin depletion impaired the ability of exendin-4, a clinically used GLP-1 analog, to reduce body weight in rats, suggesting that serotonin is a critical mediator of the energy balance impact of GLP-1 receptor (GLP-1R) activation. Serotonin turnover and expression of 5-hydroxytryptamine (5-HT) 2A (5-HT2A) and 5-HT2C serotonin receptors in the hypothalamus were altered by GLP-1R activation. We demonstrate that the 5-HT2A, but surprisingly not the 5-HT2C, receptor is critical for weight loss, anorexia, and fat mass reduction induced by central GLP-1R activation. Importantly, central 5-HT2A receptors are also required for peripherally injected liraglutide to reduce feeding and weight. Dorsal raphe (DR) harbors cell bodies of serotonin-producing neurons that supply serotonin to the hypothalamic nuclei. We show that GLP-1R stimulation in DR is sufficient to induce hypophagia and increase the electrical activity of the DR serotonin neurons. Finally, our results disassociate brain metabolic and emotionality pathways impacted by GLP-1R activation. This study identifies serotonin as a new critical neural substrate for GLP-1 impact on energy homeostasis and expands the current map of brain areas impacted by GLP-1R activation.

Signalling in the Gut Endocrine Axis

Physiology & Behavior. Feb, 2017  |  Pubmed ID: 28257936

Enteroendocrine cells are sensory cells located in the intestinal epithelium that produce a variety of hormonal regulators of gastrointestinal physiology, metabolism and food intake. They detect the absorption of a wide range of ingested nutrients via pathways that include transporter-mediated uptake, G-protein coupled receptors and ion channels. This review will describe mechanisms by which the L-cells that produce Glucagon Like Peptide-1 (GLP-1) respond to different components of ingested food, and the importance of these pathways not only for understanding physiology, but also for developing new treatments for type 2 diabetes and obesity.

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