ADGRL3 genomic variation implicated in neurogenesis and ADHD links functional effects to the incretin polypeptide GIP.

Attention deficit/hyperactivity disorder (ADHD) is the most common childhood neurodevelopmental disorder. Single nucleotide polymorphisms (SNPs) in the Adhesion G Protein-Coupled Receptor L3 (ADGRL3) gene are associated with increased susceptibility to developing ADHD worldwide. However, the effect of ADGRL3 non-synonymous SNPs (nsSNPs) on the ADGRL3 protein function is vastly unknown. Using several bioinformatics tools to evaluate the impact of mutations, we found that nsSNPs rs35106420, rs61747658, and rs734644, previously reported to be associated and in linkage with ADHD in disparate populations from the world over, are predicted as pathogenic variants. Docking analysis of rs35106420, harbored in the ADGLR3-hormone receptor domain (HRM, a common extracellular domain of the secretin-like GPCRs family), showed that HRM interacts with the Glucose-dependent insulinotropic polypeptide (GIP), part of the incretin hormones family. GIP has been linked to the pathogenesis of diabetes mellitus, and our analyses suggest a potential link to ADHD. Overall, the comprehensive application of bioinformatics tools showed that functional mutations in the ADGLR3 gene disrupt the standard and wild ADGRL3 structure, most likely affecting its metabolic regulation. Further in vitro experiments are granted to evaluate these in silico predictions of the ADGRL3-GIP interaction and dissect the complexity underlying the development of ADHD.

Early (5-Day) Onset of Diabetes Mellitus Causes Degeneration of Photoreceptor Cells, Overexpression of Incretins, and Increased Cellular Bioenergetics in Rat Retina.

The effects of early (5-day) onset of diabetes mellitus (DM) on retina ultrastructure and cellular bioenergetics were examined. The retinas of streptozotocin-induced diabetic rats were compared to those of non-diabetic rats using light and transmission electron microscopy. Tissue localization of glucagon-like-peptide-1 (GLP-1), exendin-4 (EXE-4), and catalase (CAT) in non-diabetic and diabetic rat retinas was conducted using immunohistochemistry, while the retinal and plasma concentration of GLP-1, EXE-4, and CAT were measured with ELISA. Lipid profiles and kidney and liver function markers were measured from the blood of non-diabetic and diabetic rats with an automated biochemical analyzer. Oxygen consumption was monitored using a phosphorescence analyzer, and the adenosine triphosphate (ATP) level was determined using the Enliten ATP assay kit. Blood glucose and cholesterol levels were significantly higher in diabetic rats compared to control. The number of degenerated photoreceptor cells was significantly higher in the diabetic rat retina. Tissue levels of EXE-4, GLP-1 and CAT were significantly (p = 0.002) higher in diabetic rat retina compared to non-diabetic controls. Retinal cellular respiration was 50% higher (p = 0.004) in diabetic (0.53 ± 0.16 µM O2 min-1 mg-1, n = 10) than in non-diabetic rats (0.35 ± 0.07 µM O2 min-1 mg-1, n = 11). Retinal cellular ATP was 76% higher (p = 0.077) in diabetic (205 ± 113 pmol mg-1, n = 10) than in non-diabetic rats (116 ± 99 pmol mg-1, n = 12). Thus, acute (5-day) or early onslaught of diabetes-induced hyperglycemia increased incretins and antioxidant levels and oxidative phosphorylation. All of these events could transiently preserve retinal function during the early phase of the progression of diabetes.

Functional GLP-1R antibodies identified from a synthetic GPCR-focused library demonstrate potent blood glucose control.

G protein-coupled receptors (GPCRs) are a group of seven-transmembrane receptor proteins that have proven to be successful drug targets. Antibodies are becoming an increasingly promising modality to target these receptors due to their unique properties, such as exquisite specificity, long half-life, and fewer side effects, and their improved pharmacokinetic and pharmacodynamic profiles compared to peptides and small molecules, which results from their more favorable biodistribution. To date, there are only two US Food and Drug Administration-approved GPCR antibody drugs, namely erenumab and mogamulizumab, and this highlights the challenges encountered in identifying functional antibodies against GPCRs. Utilizing Twist's precision DNA writing technologies, we have created a GPCR-focused phage display library with 1 × 1010 diversity. Specifically, we mined endogenous GPCR binding ligand and peptide sequences and incorporated these binding motifs into the heavy chain complementarity-determining region 3 in a synthetic antibody library. Glucagon-like peptide-1 receptor (GLP-1 R) is a class B GPCR that acts as the receptor for the incretin GLP-1, which is released to regulate insulin levels in response to food intake. GLP-1 R agonists have been widely used to increase insulin secretion to lower blood glucose levels for the treatment of type 1 and type 2 diabetes, whereas GLP-1 R antagonists have applications in the treatment of severe hypoglycemia associated with bariatric surgery and hyperinsulinomic hypoglycemia. Here we present the discovery and creation of both antagonistic and agonistic GLP-1 R antibodies by panning this GPCR-focused phage display library on a GLP-1 R-overexpressing Chinese hamster ovary cell line and demonstrate their in vitro and in vivo functional activity.

A Reduced Incretin Effect Mediated by the rs7903146 Variant in the TCF7L2 Gene Is an Early Marker of ß-Cell Dysfunction in Obese Youth.

OBJECTIVE: The risk genotype for the common variant rs7903146 of the transcription factor 7-like-2 (TCF7L2) gene has been found to affect the incretin response in healthy and obese adults; however, whether a similar functional defect is also present in obese adolescents remains unexplored. Herein, we examined the functional effect of the rs7903146 variant in the TCF7L2 gene on the incretin effect and determined its translational metabolic manifestation by performing deep phenotyping of the incretin system, ß-cell function relative to insulin sensitivity, the gastrointestinal-induced glucose disposal (GIGD) in obese youth with normal and impaired glucose tolerance. RESEARCH DESIGN AND METHODS: Thirty-nine obese adolescents without diabetes (median age 15 [25th, 75th percentile 14, 18] years; BMI 37 [33, 43] kg/m2) were genotyped for the rs7903146 variant of TCF7L2 and underwent a 3-h oral glucose tolerance test (OGTT) followed by an isoglycemic intravenous glucose infusion (iso-intravenous glucose tolerance test [IVGTT]) to match the plasma glucose concentrations during the OGTT and a hyperglycemic clamp with arginine stimulation. The incretin effect was measured as 100 * (AUC-SROGTT - AUC-SRiso-IVGTT) / AUC-SROGTT, where AUC-SR = area under the curve of C-peptide secretion rate. Participants were grouped into tertiles according to the percentage incretin effect (high, moderate, and low) to describe their metabolic phenotype. RESULTS: The presence of T risk allele for TCF7L2 was associated with a markedly reduced incretin effect compared with the wild-type genotype (0.3% [-7.2, 14] vs. 37.8% [12.5, 52.4], P < 0.002). When the cohort was stratified by incretin effect, the high, moderate, and low incretin effect groups did not differ with respect to anthropometric features, while the low incretin effect group exhibited higher 1-h glucose (P = 0.015) and a reduced disposition index, insulin sensitivity, and insulin clearance compared with the high incretin effect group. GIGD was reduced in the low incretin effect group (P = 0.001). The three groups did not differ with respect to intravenous glucose-induced insulin secretion and arginine response during the hyperglycemic clamp. CONCLUSIONS: A reduced incretin effect and its association with the TCF7L2 variant rs7903146 identify an early metabolic phenotype in obese youth without diabetes, featuring a higher plasma glucose peak at 1 h; lower insulin secretion, sensitivity, and clearance; and GIGD.

Brain uptake pharmacokinetics of incretin receptor agonists showing promise as Alzheimer's and Parkinson's disease therapeutics.

Among the more promising treatments proposed for Alzheimer's disease (AD) and Parkinson's disease (PD) are those reducing brain insulin resistance. The antidiabetics in the class of incretin receptor agonists (IRAs) reduce symptoms and brain pathology in animal models of AD and PD, as well as glucose utilization in AD cases and clinical symptoms in PD cases after their systemic administration. At least 9 different IRAs are showing promise as AD and PD therapeutics, but we still lack quantitative data on their relative ability to cross the blood-brain barrier (BBB) reaching the brain parenchyma. We consequently compared brain uptake pharmacokinetics of intravenous 125I-labeled IRAs in adult CD-1 mice over the course of 60 min. We tested single IRAs (exendin-4, liraglutide, lixisenatide, and semaglutide), which bind receptors for one incretin (glucagon-like peptide-1 [GLP-1]), and dual IRAs, which bind receptors for two incretins (GLP-1 and glucose-dependent insulinotropic polypeptide [GIP]), including unbranched, acylated, PEGylated, or C-terminally modified forms (Finan/Ma Peptides 17, 18, and 20 and Hölscher peptides DA3-CH and DA-JC4). The non-acylated and non-PEGylated IRAs (exendin-4, lixisenatide, Peptide 17, DA3-CH and DA-JC4) had significant rates of blood-to-brain influx (Ki), but the acylated IRAs (liraglutide, semaglutide, and Peptide 18) did not measurably cross the BBB. The brain influx of the non-acylated, non-PEGylated IRAs were not saturable up to 1 µg of these drugs and was most likely mediated by adsorptive transcytosis across brain endothelial cells, as observed for exendin-4. Of the non-acylated, non-PEGylated IRAs tested, exendin-4 and DA-JC4 were best able to cross the BBB based on their rate of brain influx, percentage reaching the brain that accumulated in brain parenchyma, and percentage of the systemic dose taken up per gram of brain tissue. Exendin-4 and DA-JC4 thus merit special attention as IRAs well-suited to enter the central nervous system (CNS), thus reaching areas pathologic in AD and PD.

Dietary Cyanidin-3-Glucoside Attenuates High-Fat-Diet-Induced Body-Weight Gain and Impairment of Glucose Tolerance in Mice via Effects on the Hepatic Hormone FGF21.

BACKGROUND: Dietary polyphenols including anthocyanins target multiple organs. OBJECTIVE: We aimed to assess the involvement of glucagon-like peptide 1 (GLP-1), leptin, insulin and fibroblast growth factor 21 (FGF21) in mediating metabolic beneficial effects of purified anthocyanin cyanidin-3-glucoside (Cy3G). METHODS: Intestinal proglucagon gene (Gcg; encoding GLP-1) and liver Fgf21 expression were assessed in 6-wk-old male C57BL-6J mice fed a low-fat-diet (LFD; 10% of energy from fat), alone or with 1.6 mg Cy3G/L in drinking water for 3 wk [experiment (Exp.) 1; n = 5/group]. Similar mice were fed the LFD or a high-fat diet (HFD; 60% energy from fat) with or without Cy3G for 20 wk. Half of the mice administered Cy3G also received 4 broad-spectrum antibiotics (ABs) in drinking water between weeks 11 and 14, for a total of 6 groups (n = 8/group). Metabolic tolerance tests were conducted between weeks 2 and 16. Relevant hormone gene expression and plasma hormone concentrations were assessed mainly at the end of 20 wk (Exp. 2). RESULTS: In Exp. 1, Cy3G administration increased ileal but not colonic Gcg level by 2-fold (P < 0.05). In Exp. 2, Cy3G attenuated HFD-induced body-weight gain (20.3% at week 16), and improved glucose tolerance (26.5% at week 15) but not insulin tolerance. Although Cy3G had no effect on glucose tolerance in LFD mice, LFD/Cy3G/AB mice showed better glucose tolerance than LFD/Cy3G mice (23%). In contrast, HFD/Cy3G/AB mice showed worse glucose tolerance compared with HFD/Cy3G mice (15%). Beneficial effects of Cy3G in HFD mice were not associated with changes in plasma leptin, insulin or GLP-1 concentrations. However, Cy3G increased hepatic Fgf21 expression in mice in Exp. 1 by 4-fold and attenuated Fgf21 overexpression in HFD mice (Exp. 2, 22%), associated with increased expression of genes that encode FGFR1 and ß-klotho (>3-fold, P < 0.05). CONCLUSIONS: Dietary Cy3G may reduce body weight and exert metabolic homeostatic effects in mice via changes in hepatic FGF21.

Incretins and microRNAs: Interactions and physiological relevance.

MicroRNAs (miRNA) are one class of the small regulatory RNAs that can impact the expression of numerous genes including incretin hormones and their G protein-coupled receptors. Incretin peptides, including GLP-1, GLP-2, and GIP, are released from the gastrointestinal tract and have an crucial role in the glucose hemostasis and pancreatic beta-cell function. These hormones and their analogs with a longer half-life, glucagon like peptide-1 receptor agonists (GLP1RA), modify the expression of miRNAs. Dipeptidyl peptidase IV (DPP-4) is an enzyme that degrades the incretin hormones and is inactivated by DPP-4 inhibitors, which are a class of compounds used in the management of type 2 diabetes. DPP-4 inhibitors may also increase or reduce the expression of miRNAs. In this review, we describe the possible interactions between miRNAs and incretin hormones and the relevance of such interactions to physiological processes and diseases.

The Potential of Hibiscus sabdariffa Linn in Inducing Glucagon-Like Peptide-1 via SGLT-1 and GLPR in DM Rats.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) hormone is an incretin hormone that is secreted in the ileum and plays a role in the pancreas to increase insulin secretion, stimulate proliferation, and prevent pancreatic ß-cell apoptosis. Currently, diabetes mellitus (DM) treatment based on GLP-1 work is being developed, for instance, from herbal plants such as Hibiscus sabdariffa Linn (H. sabdariffa). Therefore, this study aims to determine the potential of H. sabdariffa in GLP-1 secretion in the ileum and its action in pancreatic ß-cells. In addition, this study also aims to determine the active ingredients of H. sabdariffa (Hib) that interact with sodium-glucose cotransporter-1 (SGLT-1) so that it can increase GLP-1 secretion in the ileum and interact with GLP-1 receptors (GLP-1R) in the pancreas. METHOD: This experimental study used 24 experimental animals of Sprague-Dawley type (aged 8-10 weeks, weight 200-250 g) that were divided into 6 groups, namely, (i) normal (C), (ii) normal-Hib 200 (C-Hib200), (iii) normal-Hib 500 (C-Hib500), (iv) DM (C-DM), (v) DM-Hib200, and (vi) DM-Hib500. H. sabdariffa extract was given orally once a day for 5 weeks. Testing of GLP-1 levels in the ileum and pancreatic tissue was performed by enzyme-linked immunosorbent assay. The prediction of the interaction mechanism of the active substance H. sabdariffa against GLP-1 was done using molecular docking. RESULTS: There was a decrease in GLP-1 levels in the ileum of DM rats (p < 0.05). However, DM rats administered H. sabdariffa 500 mg/kg BW had GLP-1 levels that were the same as in normal rats (p > 0.05). This is due to active ingredients such as leucosin, which binds to SGLT-1. Administration of 500 mg/kg BW H. sabdariffa in DM rats resulted in GLP-1 levels in the pancreas that were the same as in normal rats (p > 0.05). In addition, the active ingredient of H. sabdariffa, delphinidin, binds to GLPR in the pancreas. CONCLUSION: The active ingredient of H. sabdariffa can increase GLP-1 secretion in the ileum and can interact with G protein-linked receptors in the pancreas.

Physiology of the Incretin Hormones, GIP and GLP-1-Regulation of Release and Posttranslational Modifications.

The focus of this article is on the analysis of the release and postrelease fate of the incretin hormones, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. Their actions are dealt with to the extent that they are linked to their secretion. For both hormones, their posttranslational processing is analyzed in detail, because of its importance for the understanding of the molecular heterogeneity of the hormones. Methods of analysis, in particular regarding measurements in plasma from in vivo experiments, are discussed in detail in relation to the molecular heterogeneity of the hormones, and the importance of the designations "total" versus "intact hormones" is explained. Both hormones are substrates for the ubiquitous enzyme, dipeptidyl peptidase-4, which inactivates the peptides with dramatic consequences for their physiological spectrum of activities. The role of endogenous and exogenous antagonists of the receptors is discussed in detail because of their importance for the elucidation of the physiology and pathophysiology of the hormones. Regarding the actual secretion, the most important factors are discussed, including gastric emptying rate and the influence of the different macronutrients. Additional factors discussed are the role of bile, paracrine regulation, the role of the microbiota, pharmaceuticals, and exercise. Finally, the secretion during pathological conditions is discussed. © 2019 American Physiological Society. Compr Physiol 9:1339-1381, 2019.

The early history of GIP 1969-2000: From enterogastrone to major metabolic hormone.

This paper describes the early history of Gastric Inhibitory Polypeptide, better referred to simply as GIP, from its isolation by purification from a crude preparation of CCK-PZ (cholecystokinin/pancreozymin) to its recognition as a key player in the pathogenesis of obesity and other metabolic disorders far removed from the enterogastrone properties by which it was originally identified. Augmentation of glucose mediated insulin release, the incretin effect, was discovered soon after GIP was first isolated and only much later was its important role in the pathogenesis of obesity, through mechanism other than insulin secretion, appreciated. Immunoassay - the only method by which the concentration of GIP was measured in plasma until quite recently - was found to be flawed and to depend upon which specific epitope of the hormone an assay detected. This was especially true if it was an amino-acid sequence specific to porcine rather than human GIP. A further confounder was the discovery that much of the GIP measured by immunoassay was its biological antagonist produced by cleavage of its two N-terminal amino-acids in the circulation by the same dipeptidyl-peptidase as de-activates GLP-1. Potential use of synthetic agonistic and antagonistic GIP analogues in therapeutics was barely alluded to before year 2000.

Gut-derived GIP activates central Rap1 to impair neural leptin sensitivity during overnutrition.

Nutrient excess, a major driver of obesity, diminishes hypothalamic responses to exogenously administered leptin, a critical hormone of energy balance. Here, we aimed to identify a physiological signal that arises from excess caloric intake and negatively controls hypothalamic leptin action. We found that deficiency of the gastric inhibitory polypeptide receptor (Gipr) for the gut-derived incretin hormone GIP protected against diet-induced neural leptin resistance. Furthermore, a centrally administered antibody that neutralizes GIPR had remarkable antiobesity effects in diet-induced obese mice, including reduced body weight and adiposity, and a decreased hypothalamic level of SOCS3, an inhibitor of leptin actions. In contrast, centrally administered GIP diminished hypothalamic sensitivity to leptin and increased hypothalamic levels of Socs3. Finally, we show that GIP increased the active form of the small GTPase Rap1 in the brain and that its activation was required for the central actions of GIP. Altogether, our results identify GIPR/Rap1 signaling in the brain as a molecular pathway linking overnutrition to the control of neural leptin actions.

Role of adipose tissue GLP-1R expression in metabolic improvement after bariatric surgery in patients with type 2 diabetes.

We aimed to explore the relationship between GLP-1 receptor (GLP-1R) expression in adipose tissue (AT) and incretin secretion, glucose homeostasis and weight loss, in patients with morbid obesity and type 2 diabetes undergoing bariatric surgery. RNA was extracted from subcutaneous (SAT) and visceral (VAT) AT biopsies from 40 patients randomized to metabolic gastric bypass, sleeve gastrectomy or greater curvature plication. Biochemical parameters, fasting plasma insulin, glucagon and area under the curve (AUC) of GLP-1 following a standard meal test were determined before and 1 year after bariatric surgery. GLP-1R expression was higher in VAT than in SAT. GLP-1R expression in VAT correlated with weight (r = -0.453, p = 0.008), waist circumference (r = -0.494, p = 0.004), plasma insulin (r = -0.466, p = 0.007), and systolic blood pressure (BP) (r = -0.410, p = 0.018). At 1 year, GLP-1R expression in VAT was negatively associated with diastolic BP (r = -0.361, p = 0.039) and, following metabolic gastric bypass, with the increase of GLP-1 AUC, (R2 = 0.46, p = 0.038). Finally, GLP-1R in AT was similar independently of diabetes outcomes and was not associated with weight loss after surgery. Thus, GLP-1R expression in AT is of limited value to predict incretin response and does not play a role in metabolic outcomes after bariatric surgery.

Incretin-based therapy for the treatment of bone fragility in diabetes mellitus.

Bone fractures are common comorbidities of type 2 diabetes mellitus (T2DM). Bone fracture incidence seems to develop due to increased risk of falls, poor bone quality and/or anti-diabetic medications. Previously, a relation between gut hormones and bone has been suspected. Most recent evidences suggest indeed that two gut hormones, namely glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), may control bone remodeling and quality. The GIP receptor is expressed in bone cells and knockout of either GIP or its receptor induces severe bone quality alterations. Similar alterations are also encountered in GLP-1 receptor knock-out animals associated with abnormal osteoclast resorption. Some GLP-1 receptor agonist (GLP-1RA) have been approved for the treatment of type 2 diabetes mellitus and although clinical trials may not have been designed to investigate bone fracture, first results suggest that GLP-1RA may not exacerbate abnormal bone quality observed in T2DM. The recent design of double and triple gut hormone agonists may also represent a suitable alternative for restoring compromised bone quality observed in T2DM. However, although most of these new molecules demonstrated weight loss action, little is known on their bone safety. The present review summarizes the most recent findings on peptide-based incretin therapy and bone physiology.

Downregulation of a GPCR by ß-Arrestin2-Mediated Switch from an Endosomal to a TGN Recycling Pathway.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in nutrient homeostasis. GIP receptor (GIPR) is constitutively internalized and returned to the plasma membrane, atypical behavior for a G-protein-coupled receptor (GPCR). GIP promotes GIPR downregulation from the plasma membrane by inhibiting recycling without affecting internalization. This transient desensitization is achieved by altered intracellular trafficking of activated GIPR. GIP stimulation induces a switch in GIPR recycling from a rapid endosomal to a slow trans-Golgi network (TGN) pathway. GPCR kinases and ß-arrestin2 are required for this switch in recycling. A coding sequence variant of GIPR, which has been associated with metabolic alterations, has altered post-activation trafficking characterized by enhanced downregulation and prolonged desensitization. Downregulation of the variant requires ß-arrestin2 targeting to the TGN but is independent of GPCR kinases. The single amino acid substitution in the variant biases the receptor to promote GIP-stimulated ß-arrestin2 recruitment without receptor phosphorylation, thereby enhancing downregulation.

GPR142 Controls Tryptophan-Induced Insulin and Incretin Hormone Secretion to Improve Glucose Metabolism.

GPR142, a putative amino acid receptor, is expressed in pancreatic islets and the gastrointestinal tract, but the ligand affinity and physiological role of this receptor remain obscure. In this study, we show that in addition to L-Tryptophan, GPR142 signaling is also activated by L-Phenylalanine but not by other naturally occurring amino acids. Furthermore, we show that Tryptophan and a synthetic GPR142 agonist increase insulin and incretin hormones and improve glucose disposal in mice in a GPR142-dependent manner. In contrast, Phenylalanine improves in vivo glucose disposal independently of GPR142. Noteworthy, refeeding-induced elevations in insulin and glucose-dependent insulinotropic polypeptide are blunted in Gpr142 null mice. In conclusion, these findings demonstrate GPR142 is a Tryptophan receptor critically required for insulin and incretin hormone regulation and suggest GPR142 agonists may be effective therapies that leverage amino acid sensing pathways for the treatment of type 2 diabetes.

The role of Gut Microbiota in the development of obesity and Diabetes.

Obesity and its associated complications like type 2 diabetes (T2D) are reaching epidemic stages. Increased food intake and lack of exercise are two main contributing factors. Recent work has been highlighting an increasingly more important role of gut microbiota in metabolic disorders. It's well known that gut microbiota plays a major role in the development of food absorption and low grade inflammation, two key processes in obesity and diabetes. This review summarizes key discoveries during the past decade that established the role of gut microbiota in the development of obesity and diabetes. It will look at the role of key metabolites mainly the short chain fatty acids (SCFA) that are produced by gut microbiota and how they impact key metabolic pathways such as insulin signalling, incretin production as well as inflammation. It will further look at the possible ways to harness the beneficial aspects of the gut microbiota to combat these metabolic disorders and reduce their impact.

Glucagon-like peptide-1: The missing link in the metabolic clock?

Circadian expression of clock genes in peripheral tissues is critical to the coordinated regulation of intestinal digestive and absorptive functions, insulin secretion, and peripheral tissue nutrient deposition during periods of nutrient ingestion, thereby preventing metabolic dysregulation. As glucagon-like peptide-1 is a key incretin hormone that regulates glucose-dependent insulin secretion, we hypothesized that this intestinal hormone is a player in the peripheral metabolic clock, linking nutrient ingestion to insulin secretion. We have now established that secretion of glucagon-like peptide-1 from the intestinal L cell shows a rhythmic pattern in rats and humans in vivo that is altered by circadian disruptors, such as constant light exposure, consumption of a Western diet and feeding at inappropriate times (i.e., during the light period in rodents). Interestingly, the alterations in the rhythm of the glucagon-like peptide-1 secretory responses were found to parallel the changes in the pattern of insulin responses in association with significant impairments in glucose tolerance. Furthermore, we have detected circadian clock gene expression, and showed circadian secretion of glucagon-like peptide-1 from both the murine and human L cell in vitro. These findings demonstrate that glucagon-like peptide-1 is a functional component of the peripheral metabolic clock, and suggest that altered release of glucagon-like peptide-1 might play a role in the metabolic perturbations that result from circadian disruption.

Altered Plasma Profile of Antioxidant Proteins as an Early Correlate of Pancreatic ß Cell Dysfunction.

Insulin resistance and ß cell dysfunction contribute to the pathogenesis of type 2 diabetes. Unlike insulin resistance, ß cell dysfunction remains difficult to predict and monitor, because of the inaccessibility of the endocrine pancreas, the integrated relationship with insulin sensitivity, and the paracrine effects of incretins. The goal of our study was to survey the plasma response to a metabolic challenge in order to identify factors predictive of ß cell dysfunction. To this end, we combined (i) the power of unbiased iTRAQ (isobaric tag for relative and absolute quantification) mass spectrometry with (ii) direct sampling of the portal vein following an intravenous glucose/arginine challenge (IVGATT) in (iii) mice with a genetic ß cell defect. By so doing, we excluded the effects of peripheral insulin sensitivity as well as those of incretins on ß cells, and focused on the first phase of insulin secretion to capture the early pathophysiology of ß cell dysfunction. We compared plasma protein profiles with ex vivo islet secretome and transcriptome analyses. We detected changes to 418 plasma proteins in vivo, and detected changes to 262 proteins ex vivo The impairment of insulin secretion was associated with greater overall changes in the plasma response to IVGATT, possibly reflecting metabolic instability. Reduced levels of proteins regulating redox state and neuronal stress markers, as well as increased levels of coagulation factors, antedated the loss of insulin secretion in diabetic mice. These results suggest that a reduced complement of antioxidants in response to a mixed secretagogue challenge is an early correlate of future ß cell failure.

Naturally-occurring TGR5 agonists modulating glucagon-like peptide-1 biosynthesis and secretion.

Selective GLP-1 secretagogues represent a novel potential therapy for type 2 diabetes mellitus. This study examined the GLP-1 secretory activity of the ethnomedicinal plant, Fagonia cretica, which is postulated to possess anti-diabetic activity. After extraction and fractionation extracts and purified compounds were tested for GLP-1 and GIP secretory activity in pGIP/neo STC-1 cells. Intracellular levels of incretin hormones and their gene expression were also determined. Crude F. cretica extracts stimulated both GLP-1 and GIP secretion, increased cellular hormone content, and upregulated gene expression of proglucagon, GIP and prohormone convertase. However, ethyl acetate partitioning significantly enriched GLP-1 secretory activity and this fraction underwent bioactivity-guided fractionation. Three isolated compounds were potent and selective GLP-1 secretagogues: quinovic acid (QA) and two QA derivatives, QA-3ß-O-ß-D-glycopyranoside and QA-3ß-O-ß-D-glucopyranosyl-(28→1)-ß-D-glucopyranosyl ester. All QA compounds activated the TGR5 receptor and increased intracellular incretin levels and gene expression. QA derivatives were more potent GLP-1 secretagogues than QA. This is the first time that QA and its naturally-occurring derivatives have been shown to activate TGR5 and stimulate GLP-1 secretion. These data provide a plausible mechanism for the ethnomedicinal use of F. cretica and may assist in the ongoing development of selective GLP-1 agonists.

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene.

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.