GIP mediates the incretin effect and glucose tolerance by dual actions on α cells and ß cells.

Glucose-dependent insulinotropic polypeptide (GIP) communicates nutrient intake from the gut to islets, enabling optimal levels of insulin secretion via the GIP receptor (GIPR) on ß cells. The GIPR is also expressed in α cells, and GIP stimulates glucagon secretion; however, the role of this action in the postprandial state is unknown. Here, we demonstrate that GIP potentiates amino acid-stimulated glucagon secretion, documenting a similar nutrient-dependent action to that described in ß cells. Moreover, we demonstrate that GIP activity in α cells contributes to insulin secretion by invoking paracrine α to ß cell communication. Last, specific loss of GIPR activity in α cells prevents glucagon secretion in response to a meal stimulus, limiting insulin secretion and driving glucose intolerance. Together, these data uncover an important axis by which GIPR activity in α cells is necessary to coordinate the optimal level of both glucagon and insulin secretion to maintain postprandial homeostasis.

Glucose and lipid effects of the ileal apical sodium-dependent bile acid transporter inhibitor GSK2330672: double-blind randomized trials with type 2 diabetes subjects taking metformin.

AIMS: To investigate the pharmacodynamics, pharmacokinetics and safety/tolerability of blocking reuptake of bile acids using the inhibitor GSK2330672 (GSK672) in patients with type 2 diabetes (T2D). METHODS: Subjects with T2D taking metformin were enrolled in two studies in which they took metformin 850 mg twice daily for 2 weeks prior to and during the randomized treatment periods. In the first crossover study (n = 15), subjects received GSK672 45 mg, escalating to 90 mg, twice daily, or placebo for 7 days. The second parallel-group study (n = 75) investigated GSK672 10-90 mg twice daily, placebo or sitagliptin for 14 days. RESULTS: In both studies, GSK672 reduced circulating bile acids and increased serum 7-α-hydroxy-4-cholesten-3-one (C4), an intermediate in the hepatic synthesis of bile acids. Compared with placebo, in the parallel-group study 90 mg GSK672 twice daily reduced fasting plasma glucose [FPG; -1.21 mmol/l; 95% confidence interval (CI) -2.14, -0.28] and weighted-mean glucose area under the curve (AUC)0-24 h (-1.33 mmol/l; 95% CI -2.30, -0.36), as well as fasting and weighted-mean insulin AUC0 -24 h . GSK672 also reduced cholesterol (LDL, non-HDL and total cholesterol) and apolipoprotein B concentrations; the maximum LDL cholesterol reduction was ∼40%. There was no change in HDL cholesterol but there was a trend towards increased fasting triglyceride levels in the GSK672 groups compared with placebo. In both studies, the most common adverse events associated with GSK672 were gastrointestinal, mostly diarrhoea (22-100%), which appeared to be independent of dose. CONCLUSIONS: In subjects with T2D on metformin, GSK672 improved glucose and lipids, but there was a high incidence of gastrointestinal adverse events.

Therapeutic potential of Takeda-G-protein-receptor-5 (TGR5) agonists. Hope or hype?

The gastrointestinal tract regulates glucose and energy metabolism, and there is increasing recognition that bile acids function as key signalling molecules in these processes. For example, bile acid changes that occur after bariatric surgery have been implicated in the effects on satiety, lipid and cholesterol regulation, glucose and energy metabolism, and the gut microbiome. In recent years, Takeda-G-protein-receptor-5 (TGR5), a bile acid receptor found in widely dispersed tissues, has been the target of significant drug discovery efforts in the hope of identifying effective treatments for metabolic diseases including type 2 diabetes, obesity, atherosclerosis, fatty liver disease and cancer. Although the benefits of targeting the TGR5 receptor are potentially great, drug development work to date has identified risks that include histopathological changes, tumorigenesis, gender differences, and questions about the translation of animal data to humans. The present article reviews the noteworthy challenges that must be addressed along the path of development of a safe and effective TGR5 agonist therapy.

GSK2374697, a long duration glucagon-like peptide-1 (GLP-1) receptor agonist, reduces postprandial circulating endogenous total GLP-1 and peptide YY in healthy subjects.

We investigated the effects of a long-duration glucagon-like peptide-1 (GLP-1) receptor agonist, GSK2374697, on postprandial endogenous total GLP-1 and peptide YY (PYY). Two cohorts of healthy subjects, one normal/overweight and one obese, were randomized to receive GSK2374697 2 mg (n = 8 each) or placebo (n = 4 and n = 2) subcutaneously on days 1, 4 and 7. Samples for plasma endogenous GLP-1 and PYY were collected after breakfast on days -1 and 12. Weighted mean area under the curve (0-4 h) of total GLP-1 and PYY in treated subjects was reduced compared with placebo. The least squares mean difference for change from baseline was -1.24 pmol/l [95% confidence interval (CI) -2.33, -0.16] and -4.47 pmol/l (95% CI -8.74, -0.20) for total GLP-1 and PYY, respectively, in normal/overweight subjects (p < 0.05 for both), and -1.56 (95% CI -2.95, -0.16) and -3.02 (95% CI -8.58, 2.55), respectively, in obese subjects (p < 0.05 for GLP-1). In healthy subjects, GSK2374697 reduced postprandial total GLP-1 and PYY levels, suggesting feedback suppression of enteroendocrine L-cell secretion of these peptides.

GSK2374697, a novel albumin-binding domain antibody (AlbudAb), extends systemic exposure of exendin-4: first study in humans--PK/PD and safety.

GSK2374697 is a genetically engineered fusion protein of a human domain antibody to exendin-4. This molecule binds with a high affinity to human serum albumin, creating a long-duration glucagon-like peptide-1 (GLP-1) receptor agonist. This study is the first evaluation of the albumin-binding domain antibody (AlbudAb) drug delivery platform in humans. The aim of this randomized clinical study was to determine the pharmacokinetics, pharmacodynamics, safety, and tolerability of GSK2374697. The pharmacokinetic profile was prolonged, with estimated half-lives ranging from 6 to 10 days. Postprandial glucose and insulin were reduced, and gastric emptying was delayed in healthy subjects, confirming anticipated GLP-1 receptor agonist pharmacology. The safety and tolerability were as expected for a potent GLP-1 agonist. Gradual titration of doses greatly improved tolerability. Rapid tolerance to nausea was observed. Study results support further investigation in type 2 diabetes and for weight loss.

Prediction of weight loss and regain following dietary, lifestyle, and pharmacologic intervention.

To develop statistical models for predicting weight loss and regain, we analyzed the phenotypic responses in an outpatient study of 60 obese subjects randomized to one of three 12-week interventions, diet (-600 kcal) alone, diet with exercise, and diet with sibutramine. This was followed by 12 weeks of observation. The best of the "baseline covariates" models was one that incorporated intervention group and baseline homeostasis model assessment-estimated insulin resistance (HOMA(IR)). It predicted week 12 weight change with R(2) of 0.38 and root mean square error (√MSE) of 2.92 kg. An alternative model incorporating baseline fat mass plus change in weight and HOMA(IR) at week 4 improved the prediction (R(2), 0.67, √MSE, 2.19 kg). We could not identify a satisfactory model to predict weight regain. We conclude that prediction of weight loss over 12 weeks is significantly improved when short-term weight change is incorporated into the model. This information could be utilized to forecast the success of a weight-loss program and to motivate and contribute to innovative designing of obesity trials.

The Biomarkers Consortium: practice and pitfalls of open-source precompetitive collaboration.

Precompetitive collaboration is a growing driver for innovation and increased productivity in biomedical science and drug development. The Biomarkers Consortium, a public-private platform for precompetitive collaboration specific to biomarkers, demonstrated that adiponectin has potential utility as a predictor of metabolic responses to peroxisome proliferator-activated receptor (PPAR) agonists in individuals with type 2 diabetes. Despite the challenges overcome by this project, the most important lesson learned is that cross-company precompetitive collaboration is a feasible robust approach to biomarker qualification.

Utility of adiponectin as a biomarker predictive of glycemic efficacy is demonstrated by collaborative pooling of data from clinical trials conducted by multiple sponsors.

This study, conducted under the Metabolic Disorders Steering Committee of the Biomarkers Consortium (a public-private partnership managed by the Foundation for the National Institutes of Health (FNIH)), analyzed blinded data on 2,688 type 2 diabetes (T2D) patients from randomized clinical trials conducted by four pharmaceutical companies. An increase in the levels of adiponectin was observed after peroxisome proliferator-activated receptor (PPAR)-agonist treatment (P < 0.0001), but not after treatment with non-PPAR drugs. This increase correlated with decreases in levels of glucose, hemoglobin A(1c) (Hb(A1c)), hematocrit, and triglycerides, and increases in levels of blood urea nitrogen, creatinine, and high-density lipoprotein cholesterol (HDL-C). Early (6-8 weeks) increases in levels of adiponectin after treatment with PPAR agonists showed a negative correlation (r = -0.21, P < 0.0001) with subsequent changes in levels of Hb(A1c). Changes in adiponectin level did not appear to be associated with baseline level of Hb(A1c). Logistic regression demonstrated that an increase in the level of adiponectin predicts a decrease in the level of Hb(A1c). These analyses confirm previously demonstrated relationships between adiponectin levels and metabolic parameters and support the robust predictive utility of adiponectin across the spectrum of glucose tolerance. Cross-company precompetitive collaboration is a feasible and powerful approach to biomarker qualification.

Right ventricular hypertrophy secondary to pulmonary hypertension is linked to rat chromosome 17: evaluation of cardiac ryanodine Ryr2 receptor as a candidate.

BACKGROUND: Fischer 344 (F344) rats are relatively resistant to hypoxia-induced right ventricular (RV) hypertrophy compared with the Wistar-Kyoto (WKY) strain. These 2 strains were used to examine the genetic basis for the differential response. METHODS AND RESULTS: Male F(2) offspring from an F344xWKY intercross were exposed to hypoxia (10% O(2)) for 3 weeks, and pulmonary artery pressure and cardiac chamber weights were measured. Genomic DNA was screened by use of polymorphic microsatellite markers across the whole genome (excluding the sex chromosomes). A quantitative trait locus (QTL) for RV weight was identified on rat chromosome 17 (lod score 6.5) that accounted for 22% of the total variance of RV weight in the F(2) population and was independent of pulmonary artery pressure. The peak was centered over marker D17Rat41, close to Chrm3, with a 1-lod support interval of 5 cM. Comparison of homologous regions in mice and humans suggested that Ryr2, the cardiac isoform of the ryanodine receptor, colocalizes with our QTL. A panel of somatic cell hybrids and fluorescence in situ hybridization mapped Ryr2 close to the gene Chrm3 within our QTL. [(3)H]Ryanodine binding to cardiac membranes from the parental strains showed a 21% reduction in B(max) in the WKY compared with the F344 strain, with no difference in K:(d). CONCLUSIONS: These data provide the first demonstration of a QTL linked to the RV response to hypoxia-induced pulmonary hypertension. The Ryr2 receptor gene lies within this QTL and merits further investigation as a candidate for this differential RV response.

Echocardiographic characterization of cardiomyopathy in Friedreich's ataxia with tissue Doppler echocardiographically derived myocardial velocity gradients.

BACKGROUND: Conventional and tissue Doppler echocardiographically derived myocardial velocity gradients (MVGs) were used to characterize the myocardium in patients with Friedreich's ataxia (FRDA), and the relationship between MVGs and the mutation in the FRDA gene, a GAA triplet repeat expansion, was investigated. METHODS AND RESULTS: We studied 29 patients with FRDA (10 men, mean age 31+/-9 years) who were homozygous for the GAA expansion in the FRDA gene and were without cardiac symptoms. A comparison was made with a group of 30 age-matched control subjects. In patients with FRDA, interventricular septal thickness (1.17+/-0.26 versus 0.85+/-0.13 cm, P:<0.005), posterior left ventricular wall thickness (1.00+/-0.24 versus 0.88+/-0.15 cm, P:<0.01), and left atrial diameter (3.3+/-0.5 versus 2.9+/-0.3 cm, P:=0.01) were increased compared with control subjects. MVGs were reduced in FRDA during systole (3.1+/-1.2 versus 4.5+/-0.5 s(-1), P:<0.0001) and in early diastole (4.9+/-2.7 versus 8.8+/-1.8 s(-1), P:<0.0001) but increased in late diastole (2.0+/-1. 3 versus 1.1+/-0.9 s(-1), P:<0.01). The strongest relationship was seen between age-corrected early diastolic MVGs and the GAA expansion in the smaller allele of the FRDA gene (r=-0.68, P:<0. 0001). CONCLUSIONS: MVGs offer a means of further characterizing the myocardial abnormalities in patients with FRDA. Early diastolic MVGs appear to relate most closely to the genetic abnormality and the consequential reduction in frataxin protein.

Genetic determination of cardiac mass in normotensive rats: results from an F344xWKY cross.

Genetic determinants affect adult cardiac mass and the predisposition to develop cardiac hypertrophy. The aim of this study was to identify quantitative trait loci (QTL) that control heart and left ventricular (LV) weight by use of normotensive inbred rat strains that differ in their adult cardiac mass phenotype. We studied 126 male F2 rats derived from a cross of normotensive Wistar-Kyoto and Fischer 344 rats. At 12 weeks of age, total heart weight and LV weight were measured. Genomic DNA from these animals was screened by use of polymorphic microsatellite markers across the whole genome (excluding the sex chromosomes). In this cross, the genetic contribution to total heart weight variation was 56%, and the genetic contribution for LV weight was 55%. Using the Mapmaker/QTL computer package, we identified a significant QTL on chromosome 3 with a log10 likelihood (LOD) score of 4.8, which accounted for 16.5% of the total variance of LV weight. This QTL was centered close to the marker D3Rat29. The QTL was also found to be significantly linked with total heart weight (LOD=4.4). These data provide the first demonstration of a QTL on chromosome 3 that plays a role in determining the difference in LV mass between normotensive Fischer 344 and Wistar- Kyoto inbred rat strains. The prostaglandin synthase 1 gene is located within the QTL.

Marked variation in the cardiomyopathy associated with Friedreich's ataxia.

OBJECTIVE: To document the cardiac phenotype associated with Friedreich's ataxia, a recessively inherited disorder characterised by spinocerebellar degeneration. SETTING: Individuals with Friedreich's ataxia who accepted the invitation to participate in the study. HYPOTHESIS: The cardiomyopathy associated with Friedreich's ataxia may offer a human model for the study of factors modulating cardiac hypertrophy. METHODS: 55 patients (mean (SD) age 30 (9) years) with a clinical diagnosis of Friedreich's ataxia were studied by clinical examination, electrocardiography, cross sectional and Doppler echocardiography, and analysis of the GAA repeat in the first intron of the frataxin gene. RESULTS: A wide variety of cardiac morphology was documented. Subjects with normal frataxin alleles had no evidence of cardiomyopathy. In homozygous subjects, a relation was found between the thickness of the interventricular septum (r = 0.53, p < 0.005), left ventricular mass (r = 0.48, p < 0.01), and the number of GAA repeats on the smaller allele of the frataxin gene. No relation was shown between the presence of electrocardiographic abnormalities (mainly repolarisation changes) and either the pattern of ventricular hypertrophy (if present) and degree of neurological disability or the length of time since diagnosis. No tendency to ventricular thinning or dilatation with age was found. Although ventricular systolic function appeared impaired in some cases, Doppler studies of ventricular filling were within the normal range for age. CONCLUSIONS: The cardiomyopathy associated with Friedreich's ataxia shows a variable phenotype which is not concordant with the presence of ECG abnormalities or the neurological features of the condition. As the genetic basis for Friedreich's ataxia has been established, further studies will help to clarify the molecular mechanisms of the cardiac hypertrophy.

Human beta-myosin heavy chain mRNA prevalence is inversely related to the degree of methylation of regulatory elements.

OBJECTIVE: Methylation of cytosine in CG dinucleotides within regulatory elements is believed to silence gene expression. These dinucleotides occur in certain important regulatory elements in the promoter region of the human beta-myosin heavy chain (beta-MHC) gene. We therefore investigated whether methylation of these elements correlates with beta-MHC gene transcription in human 'expressing' (right atrial) and 'non-expressing' (peripheral blood leucocytes) cells. METHODS: We employed 2 techniques to assess promoter methylation: (i) analysis of the susceptibility to digestion of a particular CCGG restriction site in the promoter region when genomic DNA is cleaved with the restriction endonucleases MspI (methylation-insensitive) and HpaII (methylation-sensitive), and (ii) the bisulphite-PCR method to examine in detail the methylation patterns of 3 important regulatory elements that contain CG dinucleotides. beta-MHC mRNA expression in right atrium and leucocytes was assessed using reverse-transcription-PCR with specific primers that do not detect alpha-MHC cDNA. RESULTS: The digestion pattern observed with MspI or HpaII indicated that the CCGG site was almost completely methylated in leucocytes, but relatively unmethylated in atrial myocardium from the same patients. When methylation was examined with the bisulphite-PCR method we found a reciprocal relationship between the level of beta-MHC mRNA expression in leucocytes and atrial myocardium and the degree of methylation of CG dinucleotides in the 5' regulatory elements of the gene. CONCLUSIONS: Tissue-specific methylation of the human beta-MHC gene promoter may play a role in determining the pattern of expression of this gene. Furthermore, alteration of the level of methylation may underlie the changes in transcription of this gene that occur, for example, when atrial or ventricular myocardium hypertrophies.

Downregulation of natriuretic peptide C-receptor protein in the hypertrophied ventricle of the aortovenocaval fistula rat.

OBJECTIVES: This study examined the expression of the C-type receptor for the natriuretic peptide family (NPR-C) in the ventricles of normal and aortovenocaval (AV)-fistula rats, the latter a model of cardiac volume overload producing hypertrophy of both ventricles. METHODS: Western blotting with a rabbit anti-NPR-C antibody was used to quantify NPR-C levels in ventricular membranes. NPR-C expression was localised anatomically and measured in frozen sections of cardiac tissue by histochemistry and in vitro autoradiography. RESULTS: Western blot analysis revealed a single band (approximately 120 kDa) in ventricular membranes which was reduced to approximately 60 kDa after treatment with beta-mercaptoethanol. NPR-C immunoreactivity and [125I]rat ANP1-28 binding (displaceable by the NPR-C-specific ligand C-ANP 4-23) were localised to the endocardium. NPR-C protein levels, as measured by all three techniques, were reduced significantly in the hypertrophied ventricles of AV-fistula rats compared to sham-operated animals. CONCLUSIONS: Volume-induced cardiac hypertrophy in the AV-fistula rat is associated with downregulation of endocardial NPR-C. This may be one mechanism by which the endocardium regulates the myocardial response to changes in haemodynamic load.

Angiotensin II receptor expression and inhibition in the chronically hypoxic rat lung.

1. Angiotensin II (AII) binding density and the effect of chronic AII receptor blockade were examined in the rat model of hypoxia-induced pulmonary hypertension. 2. [125I]-[Sar1,Ile2]AII binding capacity was increased in lung membranes from rats exposed to hypoxia (10% fractional inspired O2) for 7 days compared to normal rats (Bmax 108 +/- 12 vs 77 +/- 3 fmol mg-1 protein; P < 0.05), with no significant change in dissociation constant. Competition with specific AII receptor subtype antagonists demonstrated that AT1 is the predominant subtype in both normal and hypoxic lung. 3. Rats treated intravenously with the AT1 antagonist, GR138950C, 1 mg kg-1 day-1 rather than saline alone during 7 days of exposure to hypoxia developed less pulmonary hypertension (pulmonary arterial pressure: 21.3 +/- 1.7 vs 28.3 +/- 1.1 mmHg; P < 0.05), right ventricular hypertrophy (right/left ventricle weight ratio: 0.35 +/- 0.01 vs 0.45 +/- 0.01; P < 0.05) and pulmonary artery remodelling (abundance of thick-walled pulmonary vessels: 9.6 +/- 1.4% vs 20.1 +/- 0.9%; P < 0.05). 4. The reduction in cardiac hypertrophy and pulmonary remodelling with the AT1 antagonist was greater than that achieved by a dose of sodium nitroprusside (SNP) that produced a comparable attenuation of the rise in pulmonary arterial pressure during hypoxia. 5. The data suggest that AII, via the AT1 receptor, has a role in the early pathogenesis of hypoxia-induced pulmonary hypertension in the rat.

Adrenomedullin activity in chronically hypoxic rat lungs.

Adrenomedullin (AM) is a novel vasodilator with structural similarities to calcitonin gene-related peptide (CGRP). This study investigated AM activity in the rat lung during hypoxia-induced pulmonary hypertension. Both rat AM (0.2-10 nmol) and alpha-CGRP (0.2-2 nmol) produced dose-related reductions in pulmonary artery pressure in the isolated perfused lung ventilated with 2% O2. Pretreatment with alpha-CGRP, which demonstrated tachyphylaxis, or its antagonist, CGRP-(8-37), reduced the hypotensive response to AM, suggesting that part of the response to AM is mediated by CGRP receptors. 125I-labeled AM and 125I-labeled CGRP binding was significantly increased in lung membranes from 7-day hypoxic animals (AM from 1.94 +/- 0.3 to 3.36 +/- 0.4 and CGRP from 0.06 +/- 0.01 to 0.12 +/- 0.02 pmol/mg protein), with no change in dissociation constant. Moreover, the hypotensive response to both peptides was increased in the lungs of 7-day hypoxic rats. There was no significant change in lung immunoreactive AM concentrations (hypoxic 5.04 +/- 0.48 vs. control 6.28 +/- 0.76 pmol/g wet wt of tissue) or steady-state AM mRNA levels in 7-day hypoxic rats. Nonetheless, AM may be useful for the acute pharmacological manipulation of pulmonary artery pressure in hypoxia-induced pulmonary hypertension.

Hypertensive cardiac hypertrophy--is genetic variance the missing link?

1. Hypertensive cardiac hypertrophy is a major independent predictor of adverse cardiovascular events. In man the cardiac response to increased afterload is very variable, even when ambulatory blood pressure monitoring is used. Analysis of breeding experiments using normotensive and hypertensive rat strains, human twin studies and other data indicate that genetic factors play a significant role in regulating cardiac mass; in other words, a large component of total variability is accounted for by genetic variance. 2. The observation that some patients with only mild-to-moderate hypertension exhibit gross left ventricular hypertrophy (LVH) similar to the inherited hypertrophic cardiomyopathies such as familial hypertrophic cardiomyopathy (FHC) and Friedreich's ataxia (FA) has prompted us to investigate the hypothesis that genetic factors associated with excessive myocardial hypertrophy, viz. mutations in FHC and FA genes alter the hypertrophic response of the heart to pressure overload. Here we review briefly three lines of study: (i) association analysis to test whether the allele frequencies differ in hypertensive patients with or without left ventricular hypertrophy; (ii) characterization of the cardiac manifestations of FA to understand the mechanism by which the heart is affected in a disease associated with pathology in a subgroup of neurons, and (iii) creation of transgenic models to facilitate the investigation of the interaction between hypertrophic stimuli and underlying genetic predisposition. 3. Information on the nature of the cardiac-mass-modifying genes involved may be useful not only for selecting high risk patients in strategies aimed at preventing the development of LVH, but also in opening new avenues of research on the reprogramming of cardiac myocytes to encourage them to hypertrophy in situations where cardiac muscle has been damaged or is hypoplastic.