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Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with extrapancreatic effects beyond glycemic control. Here we demonstrate unexpected effects of GIP signaling in the vasculature. GIP induces the expression of the proatherogenic cytokine osteopontin (OPN) in mouse arteries via local release of endothelin-1 and activation of CREB. Infusion of GIP increases plasma OPN concentrations in healthy individuals. Plasma endothelin-1 and OPN concentrations are positively correlated in patients with critical limb ischemia. Fasting GIP concentrations are higher in individuals with a history of cardiovascular disease (myocardial infarction, stroke) when compared with control subjects. GIP receptor (GIPR) and OPN mRNA levels are higher in carotid endarterectomies from patients with symptoms (stroke, transient ischemic attacks, amaurosis fugax) than in asymptomatic patients, and expression associates with parameters that are characteristic of unstable and inflammatory plaques (increased lipid accumulation, macrophage infiltration, and reduced smooth muscle cell content). While GIPR expression is predominantly endothelial in healthy arteries from humans, mice, rats, and pigs, remarkable upregulation is observed in endothelial and smooth muscle cells upon culture conditions, yielding a "vascular disease-like" phenotype. Moreover, the common variant rs10423928 in the GIPR gene is associated with increased risk of stroke in patients with type 2 diabetes. Show less

Spinal cord injury (SCI) results in loss of movement, sensibility, and autonomic control at the level of the lesion and at lower parts of the body. Several experimental strategies have been used in attempts to increase endogenous mechanisms of neuroprotection, neuroplasticity, and repair, but with limited success. It is known that glucose-dependent insulinotropic peptide (GIP) and its receptor (GIPR) can enhance synaptic plasticity, neurogenesis, and axonal outgrowth. However, their role in the injury has never been studied. The aim of this study was to evaluate the changes in expression levels of both GIP and GIPR in acute and chronic phases of SCI in rats. Following SCI (2 to 24 h after damage), the rat spinal cord showed a lesion in which the epicenter had a cavity with hemorrhage and necrosis. Furthermore, the lesion cavity also showed ballooned cells 14 and 28 days after injury. We found that SCI induced increases in GIPR expression in areas neighboring the site of injury at 6 h and 28 days after the injury. Moreover, higher GIP expression was observed in these regions on day 28. Neuronal projections from the injury epicenter showed an increase in GIP immunoreactivity 24 h and 14 and 28 days after SCI. Interestingly, GIP was also found in progenitor cells at the spinal cord canal 24 h after injury, whereas both GIP and GIPR were present in progenitor cells at the injury epicenter 14 days after in SCI animals. These results suggest that GIP and its receptor might be implicated with neurogenesis and the repair process after SCI. Show less

Small intestinal neuroendocrine tumors (SINET) are the commonest malignancy of the small intestine; however, underlying pathogenic mechanisms remain poorly characterized. Whole-genome and -exome sequencing has demonstrated that SINETs are mutationally quiet, with the most frequent known mutation in the cyclin-dependent kinase inhibitor 1B gene (CDKN1B) occurring in only ∼8% of tumors, suggesting that alternative mechanisms may drive tumorigenesis. The aim of this study is to perform genome-wide molecular profiling of SINETs in order to identify pathogenic drivers based on molecular profiling. This study represents the largest unbiased integrated genomic, epigenomic, and transcriptomic analysis undertaken in this tumor type. Here, we present data from integrated molecular analysis of SINETs (n = 97), including whole-exome or targeted CDKN1B sequencing (n = 29), HumanMethylation450 BeadChip (Illumina) array profiling (n = 69), methylated DNA immunoprecipitation sequencing (n = 16), copy-number variance analysis (n = 47), and Whole-Genome DASL (Illumina) expression array profiling (n = 43). Based on molecular profiling, SINETs can be classified into three groups, which demonstrate significantly different progression-free survival after resection of primary tumor (not reached at 10 years vs. 56 months vs. 21 months, P = 0.04). Epimutations were found at a recurrence rate of up to 85%, and 21 epigenetically dysregulated genes were identified, including CDX1 (86%), CELSR3 (84%), FBP1 (84%), and GIPR (74%). This is the first comprehensive integrated molecular analysis of SINETs. We have demonstrated that these tumors are highly epigenetically dysregulated. Furthermore, we have identified novel molecular subtypes with significant impact on progression-free survival. Show less

The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important regulators of insulin and glucagon secretion as well as lipid metabolism and appetite. These biological functions make their respective receptors (GIPR and GLP-1R) attractive targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. The use of these native peptides in the treatment of these conditions is limited by their short half-lives. However, long-acting GLP-1R agonists and inhibitors of the enzyme that rapidly inactivates GIP and GLP-1 (dipeptidyl peptidase IV) are in clinical use. Although there is a loss of response to both hormones in T2DM, this effect appears to be more pronounced for GIP. This has made targeting GIPR less successful than GLP-1R. Furthermore, results demonstrating that GIPR knockout mice were resistant to diet-induced obesity suggested that GIPR antagonists may prove to be useful therapeutics. More recently, molecules that activate both receptors have shown promise in terms of glycemic and body weight control. This review focused on recent advances in the understanding of the signaling mechanisms and regulation of these two clinically important receptors. Show less

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 β-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 β-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 β-arrestin2 recruitment without receptor phosphorylation, thereby enhancing downregulation. Show less

Gene-by-environment (GxE) interactions determine common disease risk factors and biomedically relevant complex traits. However, quantifying how the environment modulates genetic effects on human quantitative phenotypes presents unique challenges. Environmental covariates are complex and difficult to measure and control at the organismal level, as found in GWAS and epidemiological studies. An alternative approach focuses on the cellular environment using in vitro treatments as a proxy for the organismal environment. These cellular environments simplify the organism-level environmental exposures to provide a tractable influence on subcellular phenotypes, such as gene expression. Expression quantitative trait loci (eQTL) mapping studies identified GxE interactions in response to drug treatment and pathogen exposure. However, eQTL mapping approaches are infeasible for large-scale analysis of multiple cellular environments. Recently, allele-specific expression (ASE) analysis emerged as a powerful tool to identify GxE interactions in gene expression patterns by exploiting naturally occurring environmental exposures. Here we characterized genetic effects on the transcriptional response to 50 treatments in five cell types. We discovered 1455 genes with ASE (FDR < 10%) and 215 genes with GxE interactions. We demonstrated a major role for GxE interactions in complex traits. Genes with a transcriptional response to environmental perturbations showed sevenfold higher odds of being found in GWAS. Additionally, 105 genes that indicated GxE interactions (49%) were identified by GWAS as associated with complex traits. Examples include GIPR-caffeine interaction and obesity and include LAMP3-selenium interaction and Parkinson disease. Our results demonstrate that comprehensive catalogs of GxE interactions are indispensable to thoroughly annotate genes and bridge epidemiological and genome-wide association studies. Show less

The gastro-intestinal hormone glucose-dependent insulinotropic polypeptide (GIP) potentiates glucose-induced insulin secretion, with bone anabolic effects through GIP receptor (GIPR) in animal models. We explore its potential in humans by analyzing association between polymorphisms (SNPs) in the Association between The This first exploratory association study between polymorphisms in Show less

Until very recently, G-protein dependent signal of GPCRs was thought to originate exclusively from the plasma membrane and internalized GPCRs were considered silent. Here, we demonstrated that, once internalized and located in the membrane of early endosomes, glucose-dependent Insulinotropic receptor (GIPR) continues to trigger production of cAMP and PKA activation. Direct evidence is based on identification of the active form of Gαs in early endosomes containing GIPR using a genetically encoded GFP tagged nanobody, and on detection of a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, compared to endosomes without GIP. Furthermore, decrease of the sustained phase of cAMP production and PKA activation kinetics as well as reversibility of cAMP production and PKA activity following GIP washout in cells treated with a pharmacological inhibitor of GIPR internalization, and continuous increase of cAMP level over time in the presence of dominant-negative Rab7, which causes accumulation of early endosomes in cells, were noticed. Hence the GIPR joins the few GPCRs which signal through G-proteins both at plasma membrane and on endosomes. Show less

Abnormal cannabidiol (Abn-CBD) and AS-1269574 are potent selective agonists for GPR55 and GPR119, respectively. The present study evaluated the actions and ability of these small-molecule agonists to counteract experimental diabetes in mice. Diabetes was induced in NIH Swiss mice by five consecutive daily intraperitoneal injections of 40 mg/(kg body weight) streptozotocin. Diabetic mice received daily oral administration of Abn-CBD or AS-1269574 (0.1 μmol/kg) or saline vehicle (0.9% wt/vol. NaCl) over 28 days. Body weight, food intake, fluid intake, plasma glucose, insulin, glucose tolerance, insulin release, lipid profile and pancreatic morphology were examined. Mechanism of action of agonists was assessed in acute studies using incretin-receptor-knockout mice. Abn-CBD and AS-1269574 decreased plasma glucose (20-26%, p < 0.05) and increased circulating insulin (47-48%, p < 0.05) by 10-28 days, compared with saline-treated diabetic controls. Food intake and polydipsia were reduced by both agonists (21-23%, p < 0.05 and 33-35%, p < 0.01, respectively). After 28 days of treatment, plasma glucagon concentrations were reduced (p < 0.01) and glucose tolerance was enhanced by 19-44% by Abn-CBD (p < 0.05 or p < 0.001) and AS-1269574 (p < 0.05 to p < 0.001). Plasma insulin responses were improved (p < 0.01) and insulin resistance was decreased (p < 0.05 or p < 0.01) in both Abn-CBD- and AS-1269574-treated groups. Triacylglycerols were decreased by 19% with Abn-CBD (p < 0.05) and 32% with AS-1269574 (p < 0.01) while total cholesterol was reduced by 17% (p < 0.01) and 15% (p < 0.05), respectively. Both agonists enhanced beta cell proliferation (p < 0.001) although islet area was unchanged. Acute studies in Gipr- and Glp1r-knockout mice revealed an important role for the glucagon-like peptide 1 (GLP-1) receptor in the actions of both agonists, with the glucose-lowering effects of Abn-CBD also partly mediated through the glucose-dependent insulinotropic peptide (GIP) receptor. These data highlight the potential for fatty acid G-protein-coupled receptor-based therapies as novel insulinotropic and glucose-lowering agents acting partly through the activation of incretin receptors. Show less

The gut incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) have a major role in the pathophysiology of type 2 diabetes. Specific genetic and dietary factors have been found to influence the release and action of incretins. We examined the effect of interactions between seven incretin-related genetic variants in GIPR, KCNQ1, TCF7L2 and WFS1 and dietary components (whey-containing dairy, cereal fibre, coffee and olive oil) on the risk of type 2 diabetes in the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct study. The current case-cohort study included 8086 incident type 2 diabetes cases and a representative subcohort of 11,035 participants (median follow-up: 12.5 years). Prentice-weighted Cox proportional hazard regression models were used to investigate the associations and interactions between the dietary factors and genes in relation to the risk of type 2 diabetes. An interaction (p = 0.048) between TCF7L2 variants and coffee intake was apparent, with an inverse association between coffee and type 2 diabetes present among carriers of the diabetes risk allele (T) in rs12255372 (GG: HR 0.99 [95% CI 0.97, 1.02] per cup of coffee; GT: HR 0.96 [95% CI 0.93, 0.98]); and TT: HR 0.93 [95% CI 0.88, 0.98]). In addition, an interaction (p = 0.005) between an incretin-specific genetic risk score and coffee was observed, again with a stronger inverse association with coffee in carriers with more risk alleles (0-3 risk alleles: HR 0.99 [95% CI 0.94, 1.04]; 7-10 risk alleles: HR 0.95 [95% CI 0.90, 0.99]). None of these associations were statistically significant after correction for multiple testing. Our large-scale case-cohort study provides some evidence for a possible interaction of TCF7L2 variants and an incretin-specific genetic risk score with coffee consumption in relation to the risk of type 2 diabetes. Further large-scale studies and/or meta-analyses are needed to confirm these interactions in other populations. Show less

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), glucagon (GCG) receptor (GCGR), and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide) receptor (GIPR), are three metabolically related peptide hormone receptors. A novel approach to the generation of multifunctional antibody agonists that activate these receptors has been developed. Native or engineered peptide agonists for GLP-1R, GCGR, and GIPR were fused to the N-terminus of the heavy chain or light chain of an antibody, either alone or in pairwise combinations. The fusion proteins have similar in vitro biological activities on the cognate receptors as the corresponding peptides, but circa 100-fold longer plasma half-lives. The GLP-1R mono agonist and GLP-1R/GCGR dual agonist antibodies both exhibit potent effects on glucose control and body weight reduction in mice, with the dual agonist antibody showing enhanced activity in the latter. Show less

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic β-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant β-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical. Show less

Obesity is a major worldwide health issue, with increasing prevalence in adults and children from developed and developing countries. Obesity causes several chronic diseases, including cardiovascular and respiratory diseases, osteoarthritis, hypertension, stroke, type II diabetes, obstructive sleep apnea, and several types of cancer. Previous genome-wide association studies have identified several genes associated with obesity, including LEP, LEPR, POMC, PCSK1, FTO, MC3R, MC4R, GNPDA2, TMEM18, QPCTL/GIPR, BDNF, ETV5, MAP2K5/SKOR1, SEC16B, SIM1, and TNKS/MSRA. However, most of these variants are found in the intronic or intergenic regions, making it difficult to elucidate the underlying mechanisms. Therefore, in this study, we performed a whole exome sequencing of the protein-coding regions in the total genome (exome) of two obese and one normal subject belonging to the same Thai family to identify the genes responsible for obesity. We identified 709 functional variants that were differentially expressed between obese and normal subjects; of these, 65 were predicted to be deleterious to protein structure or function. The minor allele frequency of 14 of these genes (ALOX5AP, COL9A2, DEFB126, GDPD4, HCRTR1, MLL3, OPLAH, OR4C45, PRIM2, RXFP2, TIGD6, TRPM8, USP49, and ZNF596) was low, indicating causal variants that could be associated with complex traits or diseases. Genotyping revealed HCRTR1, COL9A2, and TRPM8 to be associated with the regulation of feeding behavior and energy expenditure. These genes constituted a network of pathways, including lipid metabolism, signaling transduction, immune, membrane transport, and gene regulation pathways, and seemed to play important roles in obesity. Show less

Activation of glucose-dependent insulinotropic polypeptide receptor (GIPR) has been shown to be protective against atherosclerosis. However, effects of GIP on the heart have remained unclear. To address this question, in vitro and in vivo experiments were conducted. In isolated mouse cardiomyocytes, GIPR mRNA was detected by reverse transcription-polymerase chain reaction, and GIP stimulation increased adenosine 3',5'-cyclic monophosphate production. In apolipoprotein E-knockout mice, infusion of angiotensin II (AngII; 2,000 ng·kg(-1)·min(-1)) significantly increased the heart weights, and co-administration of GIP (25 nmol·kg(-1)·day(-1)) reversed this increase (both P<0.01). In the left ventricular walls, GIP suppressed AngII-induced cardiomyocyte hypertrophy by 34%, apoptosis by 77%, and interstitial fibrosis by 79% (all P<0.01). Furthermore, GIP reduced AngII-induced expression of transforming growth factor-β1 (TGF-β1) and hypoxia inducible factor-1α. In wild-type mice, cardiac hypertrophy was induced by AngII to a lesser extent, and prevented by GIP. In contrast, GIP did not show any cardioprotective effect against AngII-induced cardiac hypertrophy in GIPR-knockout mice. In an in vitro experiment using mouse cardiomyocytes, GIP suppressed AngII-induced mRNA expression of B-type natriuretic peptide and TGF-β1. It was demonstrated that cardiomyocytes represent a direct target of GIP action in vitro, and that GIP ameliorated AngII-induced cardiac hypertrophy via suppression of cardiomyocyte enlargement, apoptosis, and fibrosis in vivo. (Circ J 2016; 80: 1988-1997). Show less

The antidiabetic potential of thirteen novel dogfish glucagon derived analogues were assessed in vitro and in acute in vivo studies. Stable peptide analogues enhanced insulin secretion from BRIN-BD11 β-cells (p < 0.001) and reduced acute glycaemic responses following intraperitoneal glucose (25 nmol/kg) in healthy NIH Swiss mice (p < 0.05-p<0.001). The in vitro insulinotropic actions of [S2a]dogfish glucagon, [S2a]dogfish glucagon-exendin-4(31-39) and [S2a]dogfish glucagon-Lys(30)-γ-glutamyl-PAL, were blocked (p < 0.05-p<0.001) by the specific GLP-1 and glucagon receptor antagonists, exendin-4(9-39) and (desHis(1)Pro(4)Glu(9))glucagon amide but not by (Pro(3))GIP, indicating lack of GIP receptor involvement. These analogues dose-dependently stimulated cAMP production in GLP-1 and glucagon (p < 0.05-p<0.001) but not GIP-receptor transfected cells. They improved acute glycaemic and insulinotropic responses in high-fat fed diabetic mice and in wild-type C57BL/6J and GIPR-KO mice (p < 0.05-p<0.001), but not GLP-1R-KO mice, confirming action on GLP-1 but not GIP receptors. Overall, dogfish glucagon analogues have potential for diabetes therapy, exerting beneficial metabolic effects via GLP-1 and glucagon receptors. Show less

The gut incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted after meal ingestion and work in concert to promote postprandial insulin secretion and regulate glucagon secretion. GLP-1 also slows gastric emptying and suppresses appetite, whereas GIP seems to affect lipid metabolism. The introduction of selective GLP-1 receptor (GLP-1R) agonists for the treatment of type 2 diabetes and obesity has increased the scientific and clinical interest in incretins. Combining the body weight-lowering and glucose-lowering effects of GLP-1 with a more potent improvement of β cell function through additional GIP action could potentially offer a more effective treatment of diabetes and obesity, with fewer adverse effects than selective GLP-1R agonists; therefore, new drugs designed to co-activate both the GIP receptor (GIPR) and the GLP-1R simultaneously are under development. In the present review, we address advances in the field of GIPR and GLP-1R co-agonism and review in vitro studies, animal studies and human trials involving co-administration of the two incretins, as well as results from a recently developed GIPR/GLP-1R co-agonist, and highlight promising areas and challenges within the field of incretin dual agonists. Show less

Gliptins act by increasing endogenous incretin levels. Glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic peptide receptor (GIPR) are their indirect drug targets. Variants of GLP1R and GIPR have previously been associated with the incretin effect. The aim of the present pilot study was to examine associations of the GLP1R and GIPR gene variants with the glycaemic response to gliptins. A total of 140 consecutive patients with type 2 diabetes were followed-up 6 months after initiation of gliptin treatment. GLP1R rs6923761 (Gly168Ser) and GIPR rs10423928 genotyping was performed using real-time PCR, with subsequent high-resolution melting analysis. The main study outcome was reduction in glycated haemoglobin (HbA1c) after treatment. GLP1R Gly168Ser variant was significantly associated with reduction in HbA1c in an additive model (β = -0.33, p = 0.011). The mean reduction in HbA1c in Ser/Ser homozygotes was significantly lower compared with Gly-allele carriers [0.12 ± 0.23% vs. 0.80 ± 0.09% (1.3 ± 2.5 mmol/mol vs. 8.7 ± 1.0 mmol/mol); p = 0.008]. In conclusion, GLP1R missense variant was associated with a reduced response to gliptin treatment. The genotype-related effect size of ∼0.7% (8 mmol/mol) is equal to an average effect of gliptin treatment and makes this variant a candidate for use in precision medicine. Show less

The frog skin host-defence peptide hymenochirin-1B has been shown to stimulate insulin release in vitro from isolated pancreatic islets and BRIN-BD11 clonal β-cells. This study examines the effects of 28-day administration of a more potent analogue [P5K]hymenochirin-1B ([P5K]hym-1B) (75 nmol·kg(-1) body weight) to high-fat-fed mice with obesity, glucose intolerance and insulin resistance. Treatment with [P5K]hym-1B significantly decreased plasma glucose concentrations and improved glucose tolerance, insulin secretion, insulin sensitivity and increased the magnitude of the incretin effect (difference in response to oral vs intraperitoneal glucose loads). Responses to established insulin secretagogues were greater in islets isolated from treated animals compared with saline-treated controls. [P5K]hym-1B administration significantly decreased total islet area and β- and α-cell areas, and resulted in lower concentrations of circulating triglycerides and plasma and pancreatic glucagon. Peptide treatment had no effect on food intake, body weight, indirect calorimetry or circulating concentrations of amylase and marker enzymes of liver and kidney function. RT-PCR demonstrated that the Insr (insulin receptor) gene and genes involved in insulin signalling (Slc2a4, Irs1, Pik3ca, Akt1 and Pkd1) were significantly up-regulated in skeletal muscle from animals treated with [P5K]hym-1B. Expression of the Glp1r (GLP-1 receptor) and Gipr (GIP receptor) genes was significantly elevated in islets from peptide-treated mice. These data suggest that [P5K]hym-1B shows potential for development into an agent for treating patients with type 2 diabetes. Show less

The action of incretin hormones including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is potentiated in animal models defective in glucagon action. It has been reported that such animal models maintain normoglycaemia under streptozotocin (STZ)-induced beta cell damage. However, the role of GIP in regulation of glucose metabolism under a combination of glucagon deficiency and STZ-induced beta cell damage has not been fully explored. In this study, we investigated glucose metabolism in mice deficient in proglucagon-derived peptides (PGDPs)-namely glucagon gene knockout (GcgKO) mice-administered with STZ. Single high-dose STZ (200 mg/kg, hSTZ) or moderate-dose STZ for five consecutive days (50 mg/kg × 5, mSTZ) was administered to GcgKO mice. The contribution of GIP to glucose metabolism in GcgKO mice was also investigated by experiments employing dipeptidyl peptidase IV (DPP4) inhibitor (DPP4i) or Gcg-Gipr double knockout (DKO) mice. GcgKO mice developed severe diabetes by hSTZ administration despite the absence of glucagon. Administration of mSTZ decreased pancreatic insulin content to 18.8 ± 3.4 (%) in GcgKO mice, but ad libitum-fed blood glucose levels did not significantly increase. Glucose-induced insulin secretion was marginally impaired in mSTZ-treated GcgKO mice but was abolished in mSTZ-treated DKO mice. Although GcgKO mice lack GLP-1, treatment with DPP4i potentiated glucose-induced insulin secretion and ameliorated glucose intolerance in mSTZ-treated GcgKO mice, but did not increase beta cell area or significantly reduce apoptotic cells in islets. These results indicate that GIP has the potential to ameliorate glucose intolerance even under STZ-induced beta cell damage by increasing insulin secretion rather than by promoting beta cell survival. Show less

Physical activity (PA) has been shown to reduce the impact of FTO variation and obesity genetic risk scores (GRS) on BMI. We examined this interaction using a quantitative measure of PA and two adiposity indexes in a longitudinal multi-ethnic study. We analyzed the impact of PA on the association between 14 obesity predisposing variants (analyzed independently and as a GRS) and baseline/follow-up obesity measures in the multi-ethnic prospective cohort EpiDREAM (17423 participants from six ethnic groups). PA was analyzed using basic (low-moderate-high) and quantitative measures (metabolic equivalents (METS)), while BMI and the body adiposity index (BAI) were used to measure obesity. Increased PA was associated with decreased BMI/BAI at baseline/follow-up. FTO rs1421085, CDKAL1 rs2206734, TNNl3K rs1514176, GIPR rs11671664 and the GRS were associated with obesity measures at baseline and/or follow-up. Risk alleles of three SNPs displayed nominal associations with increased (NTRK2 rs1211166, BDNF rs1401635) or decreased (NPC1 rs1805081) basic PA score independently of BMI/BAI. Both basic and quantitative PA measures attenuated the association between FTO rs1421085 risk allele and BMI/BAI at baseline and follow-up. Our results show that physical activity can blunt the genetic effect of FTO rs1421085 on adiposity by 36-75% in a longitudinal multi-ethnic cohort. Show less

The glucagon-like peptide-1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor transduce nutrient-stimulated signals to control beta cell function. Although the GLP-1 receptor (GLP-1R) is a validated drug target for diabetes, the importance of the GIP receptor (GIPR) for the function of beta cells remains uncertain. We demonstrate that mice with selective ablation of GIPR in beta cells (MIP-Cre:Gipr(Flox/Flox); Gipr(-/-βCell)) exhibit lower levels of meal-stimulated insulin secretion, decreased expansion of adipose tissue mass and preservation of insulin sensitivity when compared to MIP-Cre controls. Beta cells from Gipr(-/-βCell) mice display greater sensitivity to apoptosis and markedly lower islet expression of T cell-specific transcription factor-1 (TCF1, encoded by Tcf7), a protein not previously characterized in beta cells. GIP, but not GLP-1, promotes beta cell Tcf7 expression via a cyclic adenosine monophosphate (cAMP)-independent and extracellular signal-regulated kinase (ERK)-dependent pathway. Tcf7 (in mice) or TCF7 (in humans) levels are lower in islets taken from diabetic mice and in humans with type 2 diabetes; knockdown of TCF7 in human and mouse islets impairs the cytoprotective responsiveness to GIP and enhances the magnitude of apoptotic injury, whereas restoring TCF1 levels in beta cells from Gipr(-/-βCell) mice lowers the number of apoptotic cells compared to that seen in MIP-Cre controls. Tcf7(-/-) mice show impaired insulin secretion, deterioration of glucose tolerance with either aging and/or high-fat feeding and increased sensitivity to beta cell injury relative to wild-type (WT) controls. Hence the GIPR-TCF1 axis represents a potential therapeutic target for preserving both the function and survival of vulnerable, diabetic beta cells. Show less

We have studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the novel human insulin-secreting cell line 1.1B4 configured as monolayers and pseudoislets. Incubation with the incretin gut hormones GLP-1 and GIP caused dose-dependent stimulation of insulin secretion from 1.1B4 cell monolayers and pseudoislets. The secretory responses were 1.5-2.7-fold greater than monolayers. Cell viability (MTT), DNA damage (comet assay) and apoptosis (acridine orange/ethidium bromide staining) were investigated following 2-h exposure of 1.1B4 monolayers and pseudoislets to ninhydrin, H2O2, streptozotocin, glucose, palmitate or cocktails of proinflammatory cytokines. All agents tested decreased viability and increased DNA damage and apoptosis in both 1.1B4 monolayers and pseudoislets. However, pseudoislets exhibited significantly greater resistance to cytotoxicity (1.5-2.7-fold increases in LD50) and lower levels of DNA damage (1.3-3.4-fold differences in percentage tail DNA and olive tail moment) and apoptosis (1.3-1.5-fold difference) compared to monolayers. Measurement of gene expression by reverse-transcription, real-time PCR showed that genes involved with insulin secretion (INS, PDX1, PCSK1, PCSK2, GLP1R and GIPR), cell-cell communication (GJD2, GJA1 and CDH1) and antioxidant defence (SOD1, SOD2, GPX1 and CAT) were significantly upregulated in pseudoislets compared to monolayers, whilst the expression of proapoptotic genes (NOS2, MAPK8, MAPK10 and NFKB1) showed no significant differences. In summary, these data indicate cell-communication associated with three-dimensional islet architecture is important both for effective insulin secretion and for protection of human beta cells against cytotoxicity. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone secreted in response to dietary fat and glucose. The blood GIP level is elevated in obesity and diabetes. GIP stimulates proinflammatory gene expression and impairs insulin sensitivity in cultured adipocytes. In obesity, hypoxia within adipose tissue can induce inflammation. The aims of this study were 1) to examine the proinflammatory effect of increased GIP signaling in adipose tissues in vivo and 2) to clarify the association between GIP and hypoxic signaling in adipose tissue inflammation. We administered GIP intraperitoneally to misty (lean) and db/db (obese) mice and examined adipose tissue inflammation and insulin sensitivity. We also examined the effects of GIP and hypoxia on expression of the GIP receptor (GIPR) gene and proinflammatory genes in 3T3-L1 adipocytes. GIP administration increased monocyte chemoattractant protein-1 (MCP-1) expression and macrophage infiltration into adipose tissue and increased blood glucose in db/db mice. GIPR and hypoxia-inducible factor-1α (HIF-1α) expressions were positively correlated in the adipose tissue in mice. GIPR expression increased dramatically in differentiated adipocytes. GIP treatment of adipocytes increased MCP-1 and interleukin-6 (IL-6) production. Adipocytes cultured either with RAW 264 macrophages or under hypoxia expressed more GIPR and HIF-1α, and GIP treatment increased gene expression of plasminogen activator inhibitor 1 and IL-6. HIF-1α gene silencing diminished both macrophage- and hypoxia-induced GIPR expression and GIP-induced IL-6 expression in adipocytes. Thus, increased GIP signaling plays a significant role in adipose tissue inflammation and thereby insulin resistance in obese mice, and HIF-1α may contribute to this process. Show less

Association between gastric inhibitory polypeptide receptor polymorphism, rs10423928, and body mass index in olanzapine-treated schizophrenia was examined. Body mass index change for the A/T+A/A genotypes was significantly higher than that for the T/T genotype. rs10423928 may predict weight gain in schizophrenia. Show less

High intake of carbohydrates, particularly sucrose, in western societies is associated with the development of non-alcoholic fatty liver (NAFL) and diabetes mellitus. It is unclear whether this is related primarily to the carbohydrate quantity or to the hormonal responses, particularly glucose-dependent insulinotropic polypeptide (GIP), which is released in the proximal intestine. Therefore, we investigated the role of GIP by comparing two glucose-fructose dimers, sucrose and Palatinose (isomaltulose), resorbed proximally or distally. The glycaemic and incretin responses to sucrose and Palatinose were studied by oral gavage and meal tests. We then analysed phenotypic and metabolic diet-induced changes in C57Bl/6J mice exposed to isoenergetic diets differing in carbohydrate type. Studies were repeated in GIP receptor knockout (Gipr(-/-)) mice and their wild-type littermates. Compared with sucrose, Palatinose intake resulted in slower glucose absorption and reduced postprandial insulin and GIP levels. After 22 weeks, Palatinose feeding prevented hepatic steatosis (48.5%) compared with sucrose and improved glucose tolerance, without differences in body composition and food intake. Ablation of GIP signalling in Gipr(-/-) mice completely prevented the deleterious metabolic effects of sucrose feeding. Furthermore, our microarray analysis indicated that sucrose increased 2.3-fold the hepatic expression of Socs2, which is involved in the growth hormone signalling pathway and participates in the development of NAFL. Our results suggest that the site of glucose absorption and the GIP response determine liver fat accumulation and insulin resistance. GIP may play a role in sucrose induced fatty liver by regulating the expression of Socs2. Show less

Observational studies have shown a positive association between obesity (body mass index [BMI] ≥ 30 kg/m2) and depression. Around 120 obesity-associated loci have been identified, but genetic variants associated with depression remain elusive. Recently, our team reported that the fat mass and obesity-associated (FTO) gene rs9939609 obesity-risk variant is paradoxically inversely associated with the risk of depression. This finding raises the question as to whether other obesity-associated genetic variants are also associated with depression. Twenty-one obesity gene variants other than FTO were selected from a custom ∼50,000 single-nucleotide polymorphisms (SNPs) genotyping array (ITMAT-Broad-CARe array). Associations of these 21 SNPs and an unweighted genotype score with BMI and major depressive disorder (determined using the DSM-IV diagnostic criteria) were tested in 3,209 cases and 14,195 noncases, using baseline data collected from July 2001 to August 2003 from the multiethnic EpiDREAM study. Body mass index was positively associated with depression status (odds ratio [OR] = 1.02; 95% CI, 1.02-1.03 per BMI unit; P = 2.9 × 10(-12), adjusted for age, sex, and ethnicity). Six of 21 genetic variants (rs1514176 [TNN13K], rs2206734 [CDKAL1], rs11671664 [GIPR], rs2984618 [TAL1], rs3824755 [NT5C2], and rs7903146 [TCF7L2]) and the genotype score were significantly associated with BMI (1.47 × 10(-14) ≤ P ≤ .04). Of the 21 SNPs, TAL1 rs2984618 obesity-risk allele was associated with a higher risk of major depressive disorder (P = 1.79 × 10(-4), adjusted for age, sex, BMI, and ethnicity), and BDNF rs1401635 demonstrated significant ethnic-dependent association with major depressive disorder (OR = 0.88; 95% CI, 0.80-0.97; P = .01 in non-Europeans and OR = 1.11; 95% CI, 1.02-1.20; P = .02 in Europeans; Pinteraction = 2.73 × 10(-4)). The genotype score, calculated with or without FTO rs9939609, and adjusted for the same covariates, was not associated with depression status. Our data support the view that the association between obesity and major depressive disorder at the observational level may be explained, at least in part, by shared genetic factors. Show less

Epigenetic variation has been linked to several human diseases. Proliferative diabetic retinopathy (PDR) is a major cause of vision loss in subjects with diabetes. However, studies examining the association between PDR and the genome-wide DNA methylation pattern are lacking. Our aim was to identify epigenetic modifications that associate with and predict PDR in subjects with type 1 diabetes (T1D). DNA methylation was analyzed genome-wide in 485,577 sites in blood from cases with PDR (n = 28), controls (n = 30), and in a prospective cohort (n = 7). False discovery rate analysis was used to correct the data for multiple testing. Study participants with T1D diagnosed before 30 years of age and insulin treatment within 1 year from diagnosis were selected based on 1) subjects classified as having PDR (cases) and 2) subjects with T1D who had had diabetes for at least 10 years when blood DNA was sampled and classified as having no/mild diabetic retinopathy also after an 8.7-year follow-up (controls). DNA methylation was also analyzed in a prospective cohort including seven subjects with T1D who had no/mild diabetic retinopathy when blood samples were taken, but who developed PDR within 6.3 years (converters). The retinopathy level was classified by fundus photography. We identified differential DNA methylation of 349 CpG sites representing 233 unique genes including TNF, CHI3L1 (also known as YKL-40), CHN2, GIPR, GLRA1, GPX1, AHRR, and BCOR in cases with PDR compared with controls. The majority of these sites (79 %) showed decreased DNA methylation in cases with PDR. The Natural Killer cell-mediated cytotoxicity pathway was found to be significantly (P = 0.006) enriched among differentially methylated genes in cases with PDR. We also identified differential DNA methylation of 28 CpG sites representing 17 genes (e.g. AHRR, GIPR, GLRA1, and BCOR) with P <0.05 in the prospective cohort, which is more than expected by chance (P = 0.0096). Subjects with T1D and PDR exhibit altered DNA methylation patterns in blood. Some of these epigenetic changes may predict the development of PDR, suggesting that DNA methylation may be used as a prospective marker of PDR. Show less

The stomach epithelium contains a myriad of enteroendocrine cells that modulate a range of physiological functions, including postprandial secretion of regulatory peptides, gastric motility, and nutrient absorption. Somatostatin (SST)-producing D-cells are present in the oxyntic and pyloric regions of the stomach, and provide a tonic inhibitory tone that regulates activity of neighboring enteroendocrine cells and gastric acid secretion. Cellular mechanisms underlying the effects of regulatory factors on gastric D-cells are poorly defined due to problems in identifying primary D-cells, and uncertainty remains about which stimuli influence D-cells directly. In this study, we introduce a transgenic mouse line, SST-Cre, which upon crossing with Cre reporter strains, facilitates the identification and purification of gastric D-cells, or cell-specific expression of genetically encoded calcium indicators. Populations of D-cells from the gastric antrum and corpus were isolated and analyzed by RNA sequencing and quantitative RT-PCR. The expression of hormones, hormone receptors, neurotransmitter receptors, and nutrient receptors was quantified. Pyy, Gipr, Chrm4, Calcrl, Taar1, and Casr were identified as genes that are highly enriched in D-cells compared with SST-negative cells. Hormone secretion assays performed in mixed gastric epithelial cultures confirmed that SST secretion is regulated by incretin hormones, cholecystokinin, acetylcholine, vasoactive intestinal polypeptide, calcitonin gene-related polypeptide, oligopetides, and trace amines. Cholecystokinin and oligopeptides elicited increases in intracellular calcium in single-cell imaging experiments performed using cultured D-cells. Our data provide the first transcriptomic analysis and functional characterization of gastric D-cells, and identify regulatory pathways that underlie the direct detection of stimuli by this cell type. Show less

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor β-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide. Show less

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone produced in the gastrointestinal tract that stimulates glucose dependent insulin secretion. Impaired incretin response has been documented in diabetic patients and was mainly related to the inability of the pancreatic beta cells to secrete insulin in response to GIP. Advanced Glycation End Products (AGEs) have been shown to play an important role in pancreatic beta cell dysfunction. The aim of this study is to investigate whether the exposure to AGEs can induce GIP resistance in the pancreatic beta cell line HIT-T15. Cells were cultured for 5 days in low (CTR) or high glucose (HG) concentration in the presence of AGEs (GS) to evaluate the expression of GIP receptor (GIPR), the intracellular signaling activated by GIP, and secretion of insulin in response to GIP. The results showed that incubation with GS alone altered intracellular GIP signaling and decreased insulin secretion as compared to CTR. GS in combination with HG reduced the expression of GIPR and PI3K and abrogated GIP-induced AKT phosphorylation and GIP-stimulated insulin secretion. In conclusion, we showed that treatment with GS is associated with the loss of the insulinotropic effect of GIP in hyperglycemic conditions. Show less